Non-meiotic allele introgression

ABSTRACT

Methods, uses, and compositions for manipulating genomic DNA. Some of the embodiments of the invention provide for making a founder animal that is completely free of all unplanned genetic modifications. Some embodiments are directed to removing genetic faults in established breeds without making other alterations to the genome. Other embodiments are directed to particular tools or processes such as TALENs or CRISPR with a preferred truncation. One embodiment involves introducing a targeted targeting endonuclease system and a HDR template into a cell (optionally with a mismatch in the binding of the targeting endonuclease and the targeted site). Another embodiment includes processes of making a genetically modified livestock animal comprising a genome that comprises inactivation of a neuroendocrine gene selective for sexual maturation, with the inactivation of the gene preventing the animal from becoming sexually mature. One embodiment includes compositions and methods for making livestock with a polled allele, including migrating a polled allele into a  bovine  species without changing other genes or chromosomal portions.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 16/424,356 filed May 28, 2019, which is a continuation-in-part ofU.S. application Ser. No. 15/802,272, “Efficient Non-Meiotic AlleleIntrogression” filed Nov. 2, 2017, which is a divisional of U.S.application Ser. No. 14/625,797 filed Feb. 19, 2015, which is acontinuation of U.S. patent application Ser. No. 14/263,446 filed onApr. 28, 2014, now U.S. Pat. No. 9,528,124, which claims priority toU.S. Provisional Appl. No. 61/870,401 filed on Aug. 27, 2013. U.S.application Ser. No. 16/424,356 is a continuation-in-part of U.S.application Ser. No. 13/404,662, “Genetically Modified Animals andMethods for Making the Same” filed Feb. 24, 2012, which claims priorityto U.S. Provisional Appl. No. 61/446,651 filed on Feb. 25, 2011. U.S.application Ser. No. 16/424,356 is a continuation-in-part of U.S.application Ser. No. 13/594,694, “Genetically Modified Animals andMethods for Making the Same” filed on Aug. 24, 2012, which claimspriority to U.S. Provisional Appl. No. 61/662,767 filed on Jun. 21, 2012and is a continuation-in-part of U.S. application Ser. No. 13/404,662,“Genetically Modified Animals and Methods for Making the Same”, filedFeb. 24, 2012 which claims priority to U.S. Provisional Appl. No.61/446,651 filed Feb. 25, 2011. U.S. application Ser. No. 16/424,356 isa continuation-in-part of U.S. application Ser. No. 14/067,634, “Controlof Sexual Maturation in Animals” filed on Oct. 30, 2013, which claimspriority to U.S. Provisional Appl. No. 61/870,510 filed on Aug. 27, 2013and claims priority to U.S. Provisional Appl. No. 61/720,187 filed onOct. 30, 2012. U.S. application Ser. No. 16/424,356 is acontinuation-in-part of U.S. application Ser. No. 14/154,906, “HornlessLivestock” filed on Jan. 14, 2014 which claims priority to U.S.Provisional Appl. No. 61/752,232 filed Jan. 14, 2013 and claims priorityto U.S. provisional Appl. No. 61/870,570 filed Aug. 27, 2013. Each ofthese applications is hereby incorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant number1R41HL108440-01 awarded by the National Institutes of Health, Grantnumber 1R43RR033149-01A1 awarded by the National Institutes of Healthand Biotechnology Risk Assessment Program, and competitive Grant number2012-33522-19766 awarded by the USDA—National Institute of Food andAgriculture. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 10, 2019, isnamed 53545_746_301 SL.txt and is 863,665 bytes in size.

BACKGROUND OF THE INVENTION

Animal genetic engineering has traditionally been accomplished by randominsertion of expression cassettes, which suffered from low efficiency,unpredictable expression, and/or the requirement of linked selectionmarkers. In addition, there are numerous challenges in the livestockindustry, such as the risks posed to humans by horned cattle, limitedability to control the size, weight or build of the livestock, limitedthermoregulation, etc.

SUMMARY OF THE INVENTION

Disclosed herein is a method for altering the genome of an animal cell,the method comprising: identifying a target DNA region within the animalcell, the target region comprising a target cleavage site; contactingthe animal cell with a targeted nuclease such that the nuclease cleavesthe target DNA region at the target cleavage site, wherein the targetednuclease comprises one or more binding domains that specifically bind toone or more sequences within the target DNA region. In some embodiments,the target region is from 10 nucleotides to 200 nucleotides in length,such as from 10 nucleotides to 100 nucleotides, from 10 nucleotides and75 nucleotides, from 10 to 60 nucleotides, from 10 nucleotides to 50nucleotides, from 10 to 30 nucleotides, from 30 nucleotides to 70nucleotides, from 40 nucleotides to 60 nucleotides, or from 45nucleotides to 55 nucleotides in length. The targeted nuclease can beselected from the group consisting of a transcription-activator-likeeffector nuclease (TALEN), a CRISPR-based nuclease (e.g., CRISPR/Cas9),and a zinc finger nuclease. The targeted nuclease can be atranscription-activator-like effector nuclease (TALEN). The TALEN cancomprise a first peptide and a second peptide, wherein the first peptideand the second peptide are configured to bind to one another in anon-covalent fashion, and wherein the first peptide comprises a firstbinding domain fused to a first portion of a bipartite nuclease, and thesecond peptide comprises a second binding domain fused to a secondportion of a bipartite nuclease. The bipartite nuclease can be abipartite FokI nuclease. In some embodiments, contacting the animal cellwith the targeted nuclease comprises delivering mRNA encoding the TALENinto the animal cell such that the mRNA is expressed to produce theTALEN within the cell. A nuclear localization signal can be coupled tothe TALEN.

In some embodiments, mRNA is delivered into the animal cell by any oneof: electroporation, transfection, lipofection, liposome, nucleofection,biolistic particle delivery, nanoparticle delivery, lipid transfection,electrofusion, or direct injection.

Contacting the animal cell with the targeted nuclease can compriseexpressing the targeted nuclease from plasmid DNA inside the animalcell. The targeted nuclease can be a CRISPR-based nuclease. The targetednuclease can be a zinc finger nuclease.

The method can be performed without introducing into the animal cell (1)a fluorescent marker gene or (2) a reporter gene that, when incorporatedinto chromosomal DNA of the cell, confers a trait on the cell thatpermits isolation by one or more survival selection criteria (e.g.,survival in the presence of a small molecule). The animal cell can be anartiodactyl cell. The animal cell can be a cell of a livestock animal.The livestock animal can be selected from the group consisting of swine,cows, sheep, and goats. The animal cell can be an animal cell selectedfrom the group consisting of cattle, swine, sheep, chicken, goats,rabbit, and fish. The animal cell can be a bovine cell or a porcinecell. The animal cell can be a primary somatic cell.

The method can further comprise cloning the primary somatic cell toproduce one or more embryos; and implanting the one or more embryos intoa surrogate mother. Cloning the primary somatic cell can comprisesomatic cell nuclear transfer or chromatin transfer. The method canfurther comprise producing a gene-edited animal from the implantedembryo.

The animal cell can be a totipotent or pluripotent cell. The animal cellcan be a cell from an embryo.

The method can further comprise implanting the embryo into a surrogatemother. The method can further comprise producing a gene-edited animalfrom the implanted embryo.

The targeted nuclease can cleave the target DNA region at or adjacent toa neuroendocrine gene involved in sexual maturation. The neuroendocrinegene can be selected from the group consisting of GPR54, KISS1, andGnRH11. The neuroendocrine gene of the resulting animal cell can beinactivated. Inactivation of the neuroendocrine gene can prevent naturalsexual maturation. Inactivation of the neuroendocrine gene can involveinsertion of a stop codon in a sequence of the neuroendocrine gene.

The method can further comprise administering a rescue agent to ananimal that comprises or is derived from the animal cell such that theanimal proceeds to sexual maturity. The rescue agent can comprise agonadotropin or a gonadotropin analogue. The rescue agent can comprisekisspeptin.

The method can further comprise contacting the animal cell with ahomology-dependent repair (HDR) template such that the HDR template isincorporated into genomic DNA of the animal cell, thereby altering thegenome of the animal cell. Incorporation of the HDR template into thegenomic DNA of the animal cell can result in an animal cell with anallele that is not present (or differs from the corresponding allele) inthe animal cell prior to contacting the animal cell with the HDRtemplate. Contacting the animal cell with an HDR template can compriseexpressing a vector that encodes the HDR template within the animalcell. The incorporated allele can be identical to an allele from a firstbreed that differs from a corresponding allele of a second breed fromwhich the animal cell was originally derived. The first breed can beBelgian Blue cattle and the second breed can be Wagyu cattle or Nelorecattle. The incorporated allele can be a myostatin allele that causes adouble-muscling phenotype. The animal cell, after incorporation of theHDR template, can be homozygous for the allele. The animal cell, afterincorporation of the HDR template, can be heterozygous for the allele.The allele can have an insertion or a deletion relative to acorresponding allele in the animal cell prior to contact with thetargeted nuclease and incorporation of the HDR template. The allele canhave a single nucleotide polymorphism relative to the correspondingallele in the animal cell prior to contact with the targeted nucleaseand incorporation of the HDR template. The HDR template can comprise afirst arm and a second arm, wherein the first arm is homologous to DNAon a first side of the target cleavage site and the second arm ishomologous to DNA on a second side of the target cleavage site. Thesequence of the homology-dependent repair template can be incorporatedinto the genomic DNA of the animal cell at a success rate of greaterthan 1%. The HDR template can be single-stranded DNA. The allele can bethe polled allele. Incorporation of the HDR template into the animalcell can result in a cell that comprises a natural allele that differsfrom a corresponding native allele, wherein the natural allele isselected from CWC15, ApaF1, GDF8, IGF2, SOCS2, DGAT1, GHRHR, TP53, DAZL,APC, PTEN, RB1, Smad4, BUB1B, BRCA1, BRCA2, ST14, AKT1, EGF, EGFR, KRAS,PDGFRA/B, LDLR, ApoE, ApoB, NOD2, VANGL1, VANGL2, miR-145, BMP10, SOS1,PTPN11, Nrg1, Kir6.2, GATA4, Hand2, and HLA-DQA. The targeted nucleasecan induce a double-strand break at the cleavage site.

The method can further comprise delivering a recombinase to the animalcell. The method can produce a cell. The method can produce an animal.The method can produce a descendant of the animal.

Disclosed herein is a method of modifying a bovine cell, the methodcomprising: contacting the bovine cell with a targeted endonuclease thattargets and cuts a gene encoding the prolactin receptor; contacting thebovine cell with a homology-dependent repair template such that thetemplate integrates into the genome of the bovine cell to encode atruncated prolactin receptor protein. The truncated prolactin receptorprotein can be 461 amino acids in length. The targeted endonuclease canbe selected from a zinc finger nuclease, a TAL effector nuclease (TALEN)and a CRISPR/Cas 9 nuclease. The targeted endonuclease can be a TALeffector nuclease (TALEN).

In one aspect, the TALEN has zero mismatches to a targeted region of thegene encoding the prolactin receptor. The method can further comprisecontacting the bovine cell with a targeted endonuclease comprisesexpressing exogenous mRNA encoding a TAL effector nuclease (TALEN).

Disclosed herein is a method of genetically modifying a bovine cell, themethod comprising: obtaining a bovine cell; and editing a horned gene ofthe bovine cell such that the horned gene is edited to a polled gene.The horned gene of the bovine cell can comprise a nucleotide sequenceaccording to SEQ ID NO: 385 or a nucleotide sequence that has at least95% sequence identity to SEQ ID NO: 385.

The polled gene can comprise the nucleotide sequence according to SEQ IDNO: 386 or a nucleotide sequence that has at least 95% sequence identityto SEQ ID NO: 386. The horned gene can comprise the nucleotide sequenceof SEQ ID NO: 385 and the polled gene comprises the nucleotide sequenceof SEQ ID NO: 386.

In another aspect, editing the horned gene does not involve meioticintrogression.

Editing the horned gene can comprise implementing CRISPR, zinc fingernuclease, meganuclease, or TALEN technology. Editing the horned gene cancomprise contacting the bovine cell with a TALEN that targets the hornedgene. Editing the horned gene can comprise introducing into the bovinecell a homology directed repair (HDR) template homologous to a portionof the horned gene. The TALEN can target the horned gene at a DNA targetsequence according to any of SEQ ID NOs: 240, 347, 348, 149, 150, 151,152 and 153.

In some embodiments, editing a horned gene of the bovine cell such thatthe horned gene is edited to a polled gene comprises a 202 bpinsertion-deletion event.

The HDR template can comprise a nucleotide sequence of SEQ ID NO: 381.The TALEN can comprise an amino acid sequence according to SEQ ID NOS:460-467. Editing the horned gene can comprise implementing CRISPRtechnology using guide RNA.

In one aspect the bovine cell, after editing, is heterozygous for thepolled gene. Alternatively, the bovine cell, after editing, can behomozygous for the polled gene. The bovine cell can be a somatic bovinecell. The method can further comprise transferring a nucleus of thesomatic bovine cell to an enucleated egg of the same species.

The method can further comprise producing an animal that is derived fromthe cell. The method can be used to produce a cell. The method can beused to produce an animal. The animal can comprise a polled phenotype.The method can be used to produce an animal and a descendant of theanimal.

Described herein is a non-human animal made by a method of introgressingan allele or gene into chromosomal DNA of a non-human animal cellcomprising introducing into a cell isolated from a non-human animalline: (i) a CRISPR/Cas endonuclease; (ii) a guide RNA (gRNA) comprisinga spacer RNA sequence that interacts with a target sequence in thechromosomal DNA of the cell; (iii) a homology-directed repair (HDR)template DNA sequence encoding an allele or a gene flanked by sequenceshomologous to the target sequence in a chromosomal DNA of the cell; and(iv) cloning the cell; wherein said introducing alters the chromosomalDNA of the cell to have identity with the HDR template DNA sequence atthe target sequence in the chromosomal DNA, thereby introgressing theallele or the gene into the chromosomal DNA of the cell, wherein the HDRtemplate DNA sequence also comprises a DNA sequence encoding a mismatchin the target sequence that alters the interaction with the RNA spacersequence of the gRNA, and wherein the mismatch is introduced into thechromosomal DNA of the cell and creates a sequence in the chromosomalDNA of the animal that is not found in the non-human animal line.

In one aspect, the mismatch creates a sequence in the chromosomal DNA ofthe animal that is not found in the same breed as the animal line. Themismatch can create a sequence that is not found in nature. The mismatchcan comprise a substitution of a DNA base for a base that does notpromote binding to the gRNA of a CRISPR/Cas. The substitution cancomprise a 1 to 5 base pair substitution. The mismatch can comprise aninsertion or a deletion of a DNA base. The mismatch can comprise aninsertion of 1-5 DNA bases. The mismatch can comprise a deletion of 1-5DNA bases.

In another aspect, the target sequence can encode at least a part of anendogenous allele, wherein the HDR template DNA sequence encodes anatural allele that is homologous to the endogenous allele flanked bysequences homologous to the target sequence in the chromosomal DNA ofthe animal, and wherein the natural allele replaces the endogenousallele. The target sequence can encode at least part of an endogenousallele that encodes a protein or is part of a locus associated with atrait, wherein the HDR template DNA sequence encodes a different allelethat is homologous to the endogenous allele. In some embodiments, theHDR template can encode a locus (or a part thereof) that is associatedwith an enhancement of the trait flanked by sequences homologous to thetarget sequence in the chromosomal DNA of the cell, wherein thedifferent allele replaces the endogenous allele, and wherein the traitis selected from the group consisting of: a horn growth trait, a meattrait, a meat production trait, a milk production trait, a dairy trait,and a disease resistance trait. The disease resistance trait can beselected from: a gene for resistance to African swine fever (P65/RELA):(a) genes that potential tumor growth (e.g., TP53, APC, PTEN, RB1,Smad4, BUB1B, BRCA1, BRCA2, ST14 or a combination thereof); (b) humanoncogenes for animal models of cancer (e.g., AKT1, EGF, EGFR, KRAS,PDGFRA/B or a combination thereof); (c) genes in animal models forhypercholesterolemia (to induce atherosclerosis, stroke, and Alzheimer'sdisease models), e.g., LDLR, ApoE, ApoB or a combination thereof; (d)Inflammatory Bowel disease, e.g., NOD2; (e) spina bifida, e.g., VANGL1and/or VANGL2; (f) pulmonary hypertension, e.g., miR-145; (g) genes forcardiac defects, e.g., BMP10, SOS1, PTPN11, Nrg1, Kir6.2, GATA4, Hand2,or a combination thereof and (h) celiac disease genes, e.g., HLA-DQA1.

The target sequence can encode at least part of an endogenous allele,wherein the HDR template DNA sequence encodes an allele that ishomologous to the endogenous allele flanked by sequences homologous tothe target sequence in the chromosomal DNA of the cell, and wherein theallele that is homologous to the allele replaces the endogenous allele,and wherein the allele that is homologous to the endogenous allele isfrom the same species of animal as the non-human animal line. The targetsequence can encode at least part of an endogenous allele, wherein theHDR template DNA sequence encodes an allele that is homologous to theendogenous allele flanked by sequences homologous to the target sequencein the chromosomal DNA of the cell, and wherein the allele that ishomologous to the endogenous allele replaces the endogenous allele, andwherein the allele that is homologous to the endogenous allele is notfrom the same breed of animal as the non-human animal line.

In one aspect, the cell is selected from the group consisting of aprimary cell, a primary somatic cell, a zygote, a germ cell, a stemcell, an oocyte, and a sperm. CRISPR/Cas endonuclease can be introducedinto the cell as mRNA. The cell can be homozygous for the allele or thegene introgression into the chromosomal DNA of the cell.

The non-human animal line can be selected from the group consisting of:a non-human vertebrate line, a non-human primate line, a swine line, acattle line, horse line, sheep line, a goat line, an avian line, achicken line, a rabbit line, a fish line, a dog line, and a cat line.

In another aspect, the target sequence encodes at least part of anendogenous allele, wherein the HDR template DNA sequence encodes anallele that is homologous to the endogenous allele flanked by sequenceshomologous to the target sequence in the chromosomal DNA of the cell,wherein the allele that is homologous to the endogenous allele replacesthe endogenous allele, and wherein the mismatch comprises a singlenucleotide polymorphism (SNP) that is located within the allele that ishomologous to the endogenous allele. In another aspect, the targetsequence encodes at least part of an endogenous allele, wherein the HDRtemplate DNA sequence encodes an allele that is homologous to theendogenous allele flanked by sequences homologous to the target sequencein the chromosomal DNA of the cell, wherein the allele that ishomologous to the endogenous allele replaces the endogenous allele, andwherein the mismatch consists of a SNP, that is located within theallele that is homologous to the endogenous allele. In yet anotheraspect, the target sequence encodes at least part of an endogenousallele, wherein the HDR template DNA sequence encodes an allele that ishomologous to the endogenous allele flanked by sequences homologous tothe target sequence in the chromosomal DNA of the cell, wherein theallele that is homologous to the endogenous allele replaces theendogenous allele, and wherein the mismatch comprises a plurality ofSNPs that is located within the allele that is homologous to theendogenous allele. Alternatively, the target sequence can encode atleast part of an endogenous allele, wherein the HDR template DNAsequence encodes an allele that is homologous to the endogenous alleleflanked by sequences homologous to the target sequence in thechromosomal DNA of the cell, wherein the allele that is homologous tothe endogenous allele replaces the endogenous allele, and wherein themismatch consists of a plurality of SNPs that are located within theallele that is homologous to the endogenous allele. The allele can be aSNP.

Disclosed herein is a method of making a genetically modified animal,said method comprising: (i) exposing embryos or cells to an mRNAencoding a TALEN, with the TALEN specifically binding to a targetchromosomal site in the embryos or cells, (ii) cloning the cells in asurrogate mother or implanting the embryos in a surrogate mother, withthe surrogate mother thereby gestating an animal that is geneticallymodified without a reporter gene and only at the TALEN targetedchromosomal site. In one aspect, the method includes exposing theembryos to the TALEN without a reporter gene, with more than about 1% ofthe embryos incorporating the modification at the targeted chromosomalsite. Alternatively, exposing the cells to the TALEN without a reportergene, and cloning the cells, with more than 1% of the cloned cellsproviding animals incorporating the modification at the targetedchromosomal site. The cells can be primary somatic cells or stem cells.The cells can be cloned by somatic cell nuclear transfer or chromatintransfer. The gestated animal can be homozygous for the modification.The gestated animal can be a founder animal.

The above method can be used to prepare a genetically modified animal.The animal can be a founder animal.

The genetic modification can be chosen from the group consisting of aninsertion, deletion, inversion or translocation. The TALEN can be afirst TALEN and the targeted chromosomal site is a first site, with themethod further comprising a second TALEN directed to a second targetedchromosomal site. The TALEN can be a right TALEN and further comprise aleft TALEN that is introduced with the right TALEN.

In another aspect, the method comprises providing embryos havinggenetics known to be capable of expressing a set of traits and exposingthe embryos to the TALEN without a reporter gene and screening thegestated animal for the modification and for expression of the set oftraits. Alternatively, the method comprises exposing the cells to theTALEN without a reporter gene, creating colonies of clonal cells, andtesting a subset of members of the colonies to identify coloniesincorporating the modification at the targeted chromosomal site. Testingthe subset of members of the colonies can be a destructive process. Thetesting process can be chosen from the group consisting of a nucleolyticassay, sequencing, PAGE, PCR, primer extension, or hybridization.

Alternatively, the method comprises exposing the embryos or cells tosingle stranded DNA (ssDNA) that contains an exogenous sequence, withthe genetic modification comprising the exogenous sequence. The ssDNAcan be introduced into the cell after a vector encoding a TALEN isintroduced into the cell. The ssDNA can be introduced into the cellbetween about 8 hours and about 3 days after the vector expressing aTALEN is introduced into the cell. TALEN mRNA can be directly introducedinto the cell at about the same time as the ssDNA.

The exogenous sequence can comprise an alternative allele for the TALENtargeted chromosomal site. The alternative allele can be linked to aquantitative trait or qualitative trait. Alternatively, the alternativeallele can comprise a myostatin allele present in Belgian Blue cattle.The cell or embryo can belong to a first breed and the allele can belongto a second breed of the animal. The first breed can be Wagyu or Nelorecattle and the second breed can be Belgian Blue cattle, with theoffspring being a Wagyu or Nelore calf. The allele can be chosen fromthe group consisting of an insertion, a deletion, a polymorphism, and asingle nucleotide polymorphism.

The alternative allele can provide for an enhanced livestock trait, andis chosen from the group consisting of a horn polled locus, a generecessive for fertility defects, a gene for enhancing meat production, agene for enhancing dairy production, a gene for resistance to Africanswine fever, and combinations thereof; or can provide for an animalmodel, and is chosen from the group consisting of a gene for reductionof animal size, a gene that potentiate tumor growth, an oncogene,hypercholesterolemia genes, an inflammatory bowel disease gene, a spinabifida gene, a pulmonary hypertension gene, a gene causing a cardiacdefects, and a celiac disease gene.

The targeted chromosomal site can be chosen for a disruption of a gene,wherein the disruption of the gene comprises an insertion, deletion, orsubstitution of one or more bases in a sequence encoding the gene and/ora cis-regulatory element thereof.

The genetic modification can be chosen from the group consisting of aninsertion, a deletion, a change to an exogenous nucleic acid sequence,an inversion, a translocation, a gene conversion to natural allele, agene conversion to a synthetic allele, interspecies allele migration,intraspecies allele migration, and a gene conversion to a novel allele.

The method can further comprise delivering a recombinase to the cell orembryo. The TALEN mRNA can be directly introduced into the cell as mRNA.The direct introduction into the cell can comprise a method chosen fromthe group consisting of electroporation, transfection, lipofection,liposome, nucleofection, biolistic particles, nanoparticles, lipidtransfection, electrofusion, and direct injection. The TALEN mRNA can beintroduced into the cell as a plasmid that encodes the mRNA.

In another aspect the method comprises a cell, wherein the cell is aprimary cell or stem cell and the method is performed without aselection step that requires either a positive or a negative survivalselection criterion. The cell can be chosen from the group consisting ofa livestock cell, an artiodactyl cell, a cultured cell, a primary cell,a primary somatic cell, a zygote, a primordial germ cell, a stem cell,and a zygote, or wherein the embryo is a blastocyst.

The gestated animal can be chosen from the group consisting of swine,cows, sheep, goats, chickens, rabbits, fish, zebrafish, dog, mouse, cat,mouse, rat, and laboratory animal.

Disclosed herein is a method of making a genetically modified non-humananimal cell or embryo comprising exposing embryos or cells of the animalin vitro to an mRNA encoding a TALEN, with the TALEN specificallybinding to a targeted chromosomal site in the embryos or cells, with thecells or embryos being genetically modified only at the targetedchromosomal site and with the method being performed without a reportergene. The method can further comprise culturing the cells and isolatingcolonies of the cells. The method can be performed without additivesthat create a positive or a negative selection pressure to selectgenetically modified cells. The method can comprise exposing the embryosor cells of the animal in vitro to a single stranded DNA that containsan exogenous sequence. The method can result in the production of acell.

Disclosed herein is a genetically modified animal, the animal being afounder comprising an exogenous nucleic acid sequence at an intendedsite and being free of all other genetic modifications. The exogenousnucleic acid sequence can be an allele and the intended site is ahomologue of the allele. The animal can be homozygous for the allele.

Disclosed herein is a method of creating a genetic modificationcomprising exposing a non-human primary cell in an in vitro culture or anon-human embryo to a nucleic acid encoding a TALEN, wherein the nucleicacid encodes an N-terminal leader portion having at least 80% homologyto SEQ ID NO:132. The N-terminal leader portion can have 80% homology tothe 22-residue sequence portion of SEQ ID NO:132 and a total of no morethan about 30 residues. The nucleic acid can have at least 90% homologyto SEQ ID NO: 131.

Certain embodiments are directed to hypothermic conditions for use oftargeting endonucleases. One aspect encompasses a hypothermic method oftemplate-directed repair to change a chromosomal DNA of a cell,comprising introducing into a living cell a targeted nuclease system anda nucleic acid template, wherein the targeted nuclease system and thetemplate operate to alter the chromosomal DNA to have identity to thetemplate sequence wherein the living cell is maintained at a hypothermicculturing temperature below a physiological temperature for a timeperiod of more than three days measured from the time of theintroduction. A method of hypothermic template-directed repair mayinvolve the hypothermic culturing increasing a stable incorporation ofthe template sequence into the chromosomal DNA. A method of hypothermictemplate-directed repair may further involve a culturing temperaturekept within a range from 20 to 34° C. A method of hypothermictemplate-directed repair may further involve a time period of more thanthree days. The time period may range from more than three days to abouttwo weeks. A method of hypothermic template-directed repair may furtherinvolve testing a cell for the template sequence. A method ofhypothermic template-directed repair may further involve a targetednuclease system comprising Cas9 and Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) or a plurality of TAL effector repeatsequences that are fused to the nuclease (TALEN). The targeted nucleasesystem may comprise Cas9 and Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) or a plurality of TAL effector repeatsequences that are fused to the nuclease (TALEN), wherein the nucleicacid guide is an ssDNA. A method of hypothermic template-directed repairmay further involve one or more of a nuclease, a nucleic acid guide, anda nucleic acid template introduced into the cell as an mRNA. A method ofhypothermic template-directed repair may further involve a cell selectedfrom the group consisting of a primary cell, a primary somatic cell, anegg, a sperm, a zygote, a germ cell, a stem cell, an oocyte, a sperm,and an embryo. A method of hypothermic template-directed repair mayfurther involve an animal homozygous for the template sequence.

Another aspect encompasses a method of template-directed repair tochange a chromosomal DNA of a cell, comprising introducing into a livingcell a targeted nuclease system, a nucleic acid template, and acold-factor for inhibiting cell growth, wherein the targeted nucleasesystem and the template operate to alter the chromosomal DNA to haveidentity to the template sequence. The method of template-directedrepair to change a chromosomal DNA of a cell may comprise a cold-factorfor inhibiting cell growth, such as Cold-inducible RNA-binding protein(CIRP). See Nishiyama et al., J. Cell Biol., (1997):137(4):899-908. Themethod of template-directed repair to change a chromosomal DNA of a cellmay comprise a cell-cycle inhibitor introduced by placement into aculture that comprises the cell. The cell-cycle inhibitor may beintroduced as a protein, as RNA, as an mRNA, or through a vectorencoding the cell-cycle inhibitor. The cell-cycle inhibitor may beintroduced as a protein, as RNA, as an mRNA, or through a vectorencoding the cell-cycle inhibitor wherein the template is a HDRtemplate. The template may be ssDNA. One or more of the nuclease systemand the nucleic acid template may be introduced into the cell as anmRNA. The cell may be selected from the group consisting of a primarycell, a primary somatic cell, a zygote, a germ cell, a stem cell, and anembryo. A genetically modified animal may be prepared according to themethod of any of the above. A founder animal may be made by the methodof any of the above. A cell may be made by the method of any of any ofthe above.

In another aspect, various allelic and genetic modifications arecontemplated. For example, a modification comprises a nonhuman animalcomprising a heritable exogenous allele that provides elevated fecundityand/or a heritable exogenous allele that provides parent-of-origindependent muscle hypertrophy. The animal of may be a goat. The animalmay be chosen from the group consisting of livestock, primate, swine,cattle, horse, sheep, goat, chicken, rabbit, fish, dog, mouse, cat, rat,and laboratory animal. The animal may be free of fluorescent markers,selectable markers, and expressible markers. The elevated fecundityallele of the animal may be FecB; BMPR-IB. The muscle hypertrophy alleleof the animal may be Callipyge. The animal may be homozygous for theexogenous allele.

The animal may be a non-human animal comprising an exogenous allele forAPC. The animal may comprise an allele directed to a cancerousphenotype. The exogenous allele may be a human allele. The animal may bea laboratory animal model. The animal may be selected from the groupconsisting of pig, miniature pig, Ossabow pig, rabbit, dog, sheep, andgoat. The animal may be a founder. The animal may be free of chromosomalchanges other than introgression of the exogenous allele. Disclosedherein is a method of making the animal of comprising an HDR templatedintrogression of the exogenous allele with a targeted nuclease system.The method of making the animal may comprise an HDR templatedintrogression of the exogenous allele with a targeted nuclease systemwherein the exogenous allele is chosen to be a human allele that isassociated with a cancerous phenotype.

Another aspect is an animal comprising an exogenous allele selected fromTable 7 entitled “Frequencies for recovery of colonies with HDRalleles”. Also disclosed is a method for creating the animal comprisingintrogressing an allele into an animal, the allele being chosen from thegroup listed on said Table 7 or as follows. The allele may be LDLR,e.g., for cholesterol modeling. The allele may be DAZL, e.g., forsterility. The allele may be APC, e.g., for cancer modeling. The allelemay be p53. The allele may be RAG2, e.g., knocked-out forimmunosuppression. The allele may be IL-2, e.g., knocked-out forimmunosuppression (not in Table). The allele may be a double knock-outof RAG2 and Il-2 for immunosuppression (not in Table). The allele may beROSA, e.g., for a safe harbor. The allele may be SRY, e.g., formodifications to a Y chromosome, for sex selection; —is KISS OR KISSR,e.g., for maturation or prevention thereof, e.g., knockout. The allelemay beGDF8, e.g., for increasing muscling in animals. The allele may beEIF4G, e.g., for resistance to foot and mouth diseases (FMDV). Theallele may be p65 for resistance to African Swine Fever. The allele maybecaFecB for twinning, including interspecies introgression. The allelemay be Diglyceride acyltransferase (DGAT) knockout for increased dairymerit. The allele may be ATP-binding cassette sub-family G member 2(ABCG2) for increased dairy merit. The allele may bepleiomorphic adenomagene 1 (PLAG1) for influencing age at puberty, stature and body weight.The allele may be Beta lactoglobulin for reducing allergenicity of milk,is ovomucoid, ovalbumin, ovotransferrin, or lysozyme for reducingallergenicity of avian eggs. The animal may be a pig, sheep, goat, orcow with an introgressed allele. Disclosed herein is a cell or an animalcomprising any of the above modifications. The cell or animal may be avertebrate, livestock, primate, swine, cattle, horse, sheep, goat,chicken, rabbit, fish, dog, mouse, cat, rat, or laboratory animal.

Another aspect is a method of creating a single nucleotide polymorphism(SNP) in a chromosomal DNA of a cell, comprising introducing a targetednuclease system and a HDR template into the cell, with the targetednuclease system comprising a DNA-binding member for specifically bindingan endogenous cognate sequence in the chromosomal DNA, wherein thetargeted nuclease system and the HDR template operate to alter thechromosomal DNA to have identity to the HDR template sequence, whereinthe HDR template sequence comprises a SNP. The HDR template sequence maycomprise a plurality of SNPs. The HDR template sequence may comprise anexogenous allele that replaces an endogenous allele, with the exogenousallele comprising an SNP in a sequence alignment with the endogenousallele. The HDR template sequence may comprise a plurality of SNPswherein the HDR template sequence comprises an exogenous allele thatreplaces an endogenous allele, with the exogenous allele comprising anSNP in a sequence alignment with the endogenous allele. The method mayproduce a modification wherein the chromosomal DNA is free of SNPsoutside of the exogenous allele. The method of any of the above beingfree of SNPs outside of the exogenous allele with the HDR templatesequence being identical to the chromosomal DNA except for one or moreSNPs in the exogenous allele. The method of any of the above being freeof SNPs outside of the exogenous allele with the HDR template sequencebeing identical to the chromosomal DNA except for one or more SNPs inthe exogenous allele wherein there is only one SNP. The method of any ofthe above wherein the HDR template is designed to reduce specificbinding of the DNA-binding member to the HDR template sequence and theHDR template sequence comprises a SNP, as aligned with the chromosomalDNA.

Further disclosed herein is a genetically modified animal from a firstbreed comprising an allele of a gene selected from another species oranother breed; wherein the animal of the first breed is free of geneticchanges other than the allele; methods of making the animal as set forthherein.

Another aspect of the present invention is a method of homology-directedrepair (HDR) to introgress an exogenous allele into chromosomal DNA of acell, comprising introducing a targeted endonuclease system and a HDRtemplate that comprises the exogenous allele into the cell, with thetargeted nuclease system comprising a DNA-binding member forspecifically binding an endogenous cognate sequence in the chromosomalDNA, wherein the targeted nuclease system and the HDR template operateto alter the chromosomal DNA to have identity to the HDR templatesequence to introgress the exogenous allele into the chromosomal DNA inplace of an endogenous allele, with the targeting endonuclease systemand/or HDR template comprising a feature to reduce specific binding ofthe targeting endonuclease system to DNA. The method of may comprise afeature to reduce specific binding comprising a mismatch in theDNA-binding member sequence relative to the endogenous cognate sequenceand/or a mismatch in the DNA-binding member sequence relative to the HDRtemplate sequence. The targeted endonuclease system may comprise aplurality of TAL effector repeat sequences that are fused to a nuclease(TALEN), with the TALEN comprising a sequence of Repeat VariableDiresidues (RVDs) and the mismatch is in the sequence of RVDs relativeto the endogenous cognate sequence. The targeted nuclease system maycomprise a Cas9 nuclease and a guide RNA, with the mismatch being in thegRNA sequence relative to the endogenous cognate sequence. The targetedendonuclease system may comprise a plurality of TAL effector repeatsequences that are fused to a nuclease (TALEN), with the TALENcomprising a sequence of Repeat Variable Diresidues (RVDs) and themismatch is in the sequence of RVDs relative to the HDR templatesequence. The targeted nuclease system may comprise a Cas9 nuclease anda guide RNA, with the mismatch being in the gRNA relative to the HDRtemplate sequence. The exogenous allele may be a natural allele and theHDR template may comprise the mismatch, with the mismatch creating asequence that is not found in nature. The exogenous allele may be freeof mismatches and comprise DNA expressed by the cell. The exogenousallele may comprise the mismatch and DNA expressed by the cell. Themethod may further comprise selecting the DNA-binding member sequenceand the endogenous cognate sequence so that altering the chromosomal DNAto have identity to the HDR template sequence creates the mismatch inthe DNA-binding member sequence relative to the altered chromosomal DNAsequence. The exogenous allele may be a natural allele and the HDRtemplate consists of the natural allele and DNA that has an identitywith the chromosomal DNA sequence. Selecting the DNA-binding membersequence and the endogenous cognate sequence may further compriseplacing a second mismatch in the endogenous cognate sequence that is notchanged when the chromosomal DNA is altered to have identity to the HDRtemplate. The method may further comprise selecting the DNA-bindingmember sequence and the endogenous cognate sequence to place themismatch in the endogenous cognate sequence relative to the DNA-bindingsequence, and altering the chromosomal DNA to have identity to the HDRtemplate sequence does not remove the mismatch. The mismatch maycomprise an insertion, a deletion, or a substitution. The insertion,deletion, or substitution may have a length from 1 to 20 residues. Theinsertion, deletion, or substitution may have a length from 1 to 20residues. The mismatch may be one SNP. The method may comprise aplurality of mismatches. The targeting endonuclease system may comprisea pair of TALENs that localize to the chromosomal DNA with a spacersequence between the pair, wherein the feature comprises selecting theHDR template to create a change in a length of the spacer sequence toblock cleavage of the DNA by the TALENs pair. The spacer length may bedecreased by a deletion or increased by an insertion. The spacer lengthmay be increased or decreased by a number of residues in a range from 1to 60. The cell may be selected from the group consisting of a primarycell, a primary somatic cell, a zygote, a germ cell, a stem cell, anoocyte, a sperm, and an embryo. The HDR template may be a ssDNA. Thenuclease system may be introduced into the cell as an mRNA. The targetednuclease system may specifically bind the endogenous cognate sequencewith a binding protein. The exogenous allele may comprise an APC allele.The method of any of the above may be free of reporters, fluorescentmarkers, selectable markers, and expressible markers. The cell may be alivestock cell. The cell may be from vertebrate, livestock, primate,swine, cattle, horse, sheep, goat, chicken, rabbit, fish, dog, mouse,cat, rat, and laboratory animal. The animal may be homozygous for theexogenous allele. Disclosed herein is a method of making a geneticallymodified animal comprising cloning a cell modified by the method of anyof the above. The animal may be a founder. Disclosed herein is agenetically modified animal prepared according to the method of any ofthe above. The genetically modified animal may be a founder animal.Disclosed herein is a cell made by the method of any of the above.Disclosed here in is a kit comprising the targeted nuclease system andthe HDR template of any of the above. Disclosed herein is a use of anyof the above comprising preparing a cell for research in vitro, orpreparing a cell for use in making an animal.

Another aspect comprises a genetically modified animal, the animalbelonging to a breed having an endogenous allele in the chromosomal DNAof the animal, the animal comprising a change at an SNP, the SNP beingin the endogenous allele relative to an exogenous allele found inanother species or another breed of animal. The genetically modifiedanimal may belong to a breed having an endogenous allele in thechromosomal DNA of the animal, the animal comprising an exogenous allelefound in another species or another breed of animal, with the exogenousallele having a change at an SNP relative to the endogenous allele. Inother words, the modified animal has an SNP so that it now has an allelethat is not normally found in its breed, with that allele being fromsome other breed or species. The change could be only that SNP or therecould be other changes, with the SNP being necessary to mirror thedesired allele. The SNP is not a result of random processes, but is anintended result. The animal may comprise a plurality of the SNPs. Theanimal may comprise further changes in the chromosomal DNA of the animalrelative to the exogenous allele. The animal of any of the above beingfree or reporters. The animal of any of the above being homozygous forthe SNP and/or the SNPs. The animal of any of the above being fromvertebrate, livestock, primate, swine, cattle, horse, sheep, goat,chicken, rabbit, fish, dog, mouse, cat, rat, and laboratory animal.

Another aspect comprises a method of creating a landing pad in achromosomal DNA of a cell, comprising introducing a targeted nucleasesystem and a HDR template into the cell, with the targeted nucleasesystem comprising a DNA-binding member for specifically binding anendogenous cognate sequence in the chromosomal DNA, wherein the targetednuclease system and the HDR template operate to alter the chromosomalDNA to have identity to the HDR template sequence, wherein the HDRtemplate sequence comprises a landing pad.

Also disclosed herein is a genetically modified livestock animalcomprising a genome that comprises inactivation of a neuroendocrine geneselective for sexual maturation, with the inactivation of the genepreventing the animal from becoming sexually mature. Inactivation of thegene may comprise an insertion, deletion, or substitution of one or morebases in a sequence encoding the sexual maturation gene and/or acis-regulatory element thereof. The inactivated gene may be inactivatedby: removal of at least a portion of the gene from a genome of theanimal, alteration of the gene to prevent expression of a functionalfactor encoded by the gene, or a trans-acting factor. The gene may beinactivated by the trans-acting factor, said trans-acting factor beingchosen from the group consisting of interfering RNA and a dominantnegative factor, with said trans-acting factor being expressed by anexogenous gene or an endogenous gene. The trans-acting factor maycomprise a dominant negative for GPR54. Inactivation of the gene may beunder control of an inducible system. The inducible system may comprisea member of the group consisting of Tet-On, Tet-Off, Cre-lox, and Hif1alpha. The animal may be chosen from the group consisting of cattle,swine, sheep, chicken, goats, and fish. Further disclosed is a livestockanimal of any of the above wherein the sexual maturation gene is chosenfrom the group consisting of Gpr54, Kiss1, and GnRH11. The livestockanimal may further express a trait as a result of expression of arecombinant protein. The livestock animal may express an exogenousrecombinant protein. The trait may be chosen from the group consistingof production traits, type traits, and workability traits. The livestockanimal of any of the above may be sexually immature at an age that awild type animal of the same species is sexually mature. The livestockanimal of any of the above may be genetically unable to mature without atreatment.

Further disclosed herein is a genetically modified livestock animalcomprising a genome that is heterozygous for an inactivation of aneuroendocrine gene selective for sexual maturation, wherein progenyhomozygous for the inactivated gene are thereby prevented from becomingsexually mature. The sexual maturation gene may be chosen from the groupconsisting of Gpr54, Kiss 1, and GnRH11.

Another aspect comprises an in vitro organism chosen from the groupconsisting of a cell or an embryo, the in vitro organism comprising agenome that comprises an inactivation of a sexual maturation gene. Theorganism may be a cell or embryo chosen from the group consisting ofcattle, swine, sheep, chicken, goats, rabbit, and fish. The inactivationmay be in a gene chosen from the group consisting of Gpr54, KiSS1, andGnRH11.

Another aspect comprises a process of making a livestock animalcomprising introducing, into an organism chosen from the groupconsisting of a livestock cell and a livestock embryo, an agent thatspecifically binds to a chromosomal target site of the cell and causes adouble-stranded DNA break to inactivate a neuroendocrine gene selectivefor sexual maturation, with the agent being chosen from the groupconsisting of a TALEN, a zinc finger nuclease, Cas9/CRISPR and arecombinase fusion protein. The agent may be a TALEN of a TALEN pairthat comprises a sequence to specifically bind the chromosomal targetsite, and creates the double stranded break in the gene or creates thedouble stranded break in the chromosome in combination with a furtherTALEN that creates a second double stranded break with at least aportion of the gene being disposed between the first break and thesecond break. The process may further comprise co-introducing arecombinase into the organism with the TALEN or TALENs. A transgeneexpressing the agent may be placed in a genome of the organismIntroducing the agent into an organism may comprise a method chosen fromthe group consisting of direct injection of the agent as peptides,injection of mRNA encoding the agent, exposing the organism to a vectorencoding the agent, and introducing a plasmid encoding the agent intothe organism. Further disclosed is the process of any of the abovewherein the agent is the recombinase fusion protein, with the processcomprising introducing a targeting nucleic acid sequence with the fusionprotein, with the targeting nucleic acid sequence forming a filamentwith the recombinase for specific binding to the chromosomal site. Therecombinase fusion protein may comprise a recombinase and Gal4. Theprocess of any of the above may further comprise introducing a nucleicacid into the organism, wherein the nucleic acid is inserted into thegenome of the organism at a site of the double-stranded break or betweenthe first break and second break. The process of any of the above mayfurther comprise introducing an exogenous nucleic acid template having asequence into the organism, with the genome of the organism at a site ofthe double-stranded break receiving the sequence. The exogenous templatecan be copied or actually inserted into the genome, with the resultbeing the same, regardless of the theories about it being one or theother mechanism. The result may be that the genome has the sequence ofthe template. The nucleic acid may comprise a member of the groupconsisting of a stop codon, a reporter gene, and a reporter genecassette. The process of any of the above may further comprise cloningthe animal from the organism. The animal may be chosen from the groupconsisting of cattle, swine, sheep, chicken, goats, rabbit, and fish.The sexual maturation gene may be chosen from the group consisting ofGpr54, Kiss1, and GnRH11. Inactivation of the gene may be under controlof an inducible system.

Disclosed herein is a process of raising a livestock animal comprisingadministering an agent to an animal for sexual maturation of the animal,with the agent compensating for a genetic inability of the animal tosexually mature. The agent may comprise a gonadotropin or a gonadotropinanalogue. The process may further comprise breeding the sexually matureanimal to produce progeny. The genetic inability of the animal to maturemay be a result of a genetically inactivated neuroendocrine geneselective for sexual maturation, hereafter variation 1. The inactivatedgene may be chosen from the group consisting of Gpr54, Kiss1, andGnRH11. The inactivated gene may be inactivated by: removal of at leasta portion of the gene from a genome of the animal, alteration of thegene to prevent expression of a functional factor encoded by the gene,or a trans-acting factor. The animal may be chosen from the groupconsisting of cattle, swine, chicken, sheep, fish, rabbit, and goats.The administration of the agent to the animals may take place in atreatment facility. The progeny may be distributed from the treatmentfacility to a plurality of locations to be raised.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference,including U.S. application Ser. No. 14/154,906 “Hornless Livestock,”U.S. Prov. Appl. No. 61/870,570 “Hornless Livestock”, U.S. Prov. Appl.No. 61/752,232 “Hornless Livestock”, U.S. application Ser. No.13/594,694 “Genetically Modified Animals and Methods for Making theSame,” U.S. Prov. Appl. No. 61/662,767, U.S. Prov. Appl. No. 61/446,651,U.S. application Ser. No. 13/404,662, U.S. Prov. Appl. No. 61/870,510,U.S. Prov. Appl. No. 61/720,187, and Ser. No. 14/067,634 “Cells withModified Neuroendocrine Genes.”

BRIEF DESCRIPTION OF THE DRAWINGS

Various features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1: An illustration of a TALEN and genetic modifications caused bythe same.

FIG. 2: An illustration of TALENs operating at a plurality of DNA loci.

FIG. 3A: TALEN activity in bovine embryos. An experimental overview isgiven. TALENs are designed to opposing strands of the DNA target suchthat the FokI nuclease homodimeric monomers are able to dimerize andcleave DNA between the two monomers. Bovine in vitro-produced zygotesare injected with TALEN mRNA on day 1 (D1) and cultured in vitro toblastocyst formation. Individual blastocysts (blasts) are collected onday 8, subjected to whole genome amplification (WGA) and analyzed forindels by PCR amplification and Cel-I (SURVEYOR Nuclease, Transgenomics)treatment.

FIG. 3B: TALEN activity in bovine embryos. SURVEYOR Nuclease treatmentfor analysis of indels in bovine embryos mediated by ACAN12 TALENs. Theamplicon length and predicted SURVEYOR cleavage products that areindicative of indels, is shown above.

FIG. 4: Deletions and insertions sequenced from bovine embryos treatedwith ACAN12 TALENs. The wild-type sequence is shown with TALEN bindingsites underlined. Both deletion and insertion events are identified.

FIG. 5: Comparison of TALEN scaffold for gene editing in livestockfibroblasts. Panel a) A diagram of TALEN scaffolds tested in thisexperiment. Each scaffold (+231, Christian et. al. 2010 and Carlson +63,(compare to: Miller et. al. 2011)) contains a SV40 nuclear localizationsignal (NLS) and has a C-terminal fusion of the FokI homodimer domain.Numbering is relative to the DNA binding domain. The amino acid prior tothe first repeat variable diresidue repeat (RVD) is labeled “−1” and theamino acid following the last RVD repeat is labeled “+1”. Panel b) TheSURVEYOR assay was conducted on fibroblasts transfected with eitherDMDE7.1 or ACAN12 TALEN pairs. Scaffold and temperature treatment isindicated above the gel and percent NHEJ is indicated below.Abbreviations, NT=not treated. Panel c) Activity of four additionalTALEN pairs with either the +231 or Carlson +63 scaffold.

FIG. 6: Deletions and insertions sequenced from cells treated withACAN12 TALENs are shown in SEQ ID NO: 27-38. The wild-type ACAN12sequence (SEQ ID NO. 26) is displayed in italics and the left and right(complimentary) TALEN-recognition sequences are underlined. Insertednucleotides are highlighted in boxes and mismatch nucleotides aredenoted by lower-case text.

FIG. 7: Transposon co-selection for indel enrichment. An experimentaltimeline is shown in panel (a). Day zero (D0), cells are transfectedwith a mixture of plasmids including an expression cassette for eachTALEN, a transposon encoding a selection marker, and atransposase-expression cassette. The TALEN plasmid is the majorcomponent (4-fold excess by mass) of each transfection. Fibroblasts werecultured in DMEM (high glucose) supplemented to 10% FBS, 20 mm GlutaMAXand 1× Penn/Strep solution (all from Invitrogen) and transfected byusing the Basic Fibroblast Nuclofection Kit (Amaxa Biosystems/Lonza) orMinis LT1 reagent (Minis) as previously described (Carlson 2011).Briefly, each transfection included 500,000-1,000,000 fibroblasts, 2 ugeach TALEN plasmid and 750 ng of transposon components (500 ngpKT2P-PTK; 200 ng pKC-SB100X; 50 ng pMAX-EGFP (Lonza)). Transfectedcells are cultured for 3 days at either 30 or 37 degrees Celsius priorto splitting, collection of a sample for SURVEYOR assay and re-platingfor extended culture +/− selection for transposon integration. All cellsare cultured at 37 degrees Celsius after day 3. Cells cultured for 14+days are collected for SURVEYOR assay and cryopreserved for downstreamapplications, e.g., single-cell nuclear transfer. Panel b) Fibroblastswere transfected using cationic-lipids. No activity was observed at day3 (due to low transfection efficiency) so only data for day 14+populations is provided. Temperature treatment, selection, and TALEN id(identified by letters A-C as indicated in panel (c)) are shown abovethe gel. Panel c) Fibroblasts were transfected by Nucleofection andpercent NHEJ was measured at day 3, and in day 14+ non-selected (NS) andselected (S) populations. Temperature treatment is indicated above eachmatrix. Abbreviations: nd=not detected; wt=wild type amplicon, SURVEYORtreated.

FIG. 8A: Direct PCR sequencing for identification of indels. PCRamplicons from individual fibroblast colonies were purified, sequencedand compared to the wild-type sequence SEQ ID NO: 68. Mutation of oneallele or non-overlapping mutations of both alleles will result indouble sequence near the TALEN recognition sites (top). Overlappingbi-allelic mutations can be identified where differences between eachallele can be identified by double peaks flanking the mutation site.Colonies with homozygous mutations do not display double peaks near theindel site.

FIG. 8B: Sequence comparisons of wild-type and bi-allelic clones withhomozygous indels, as in FIG. 8A.

FIG. 9A: DMD (Duchenne's Muscular Dystrophy) Bi-allelic modificationalleles. Colonies with either homozygous modification alleles (i.e.,both alleles harbor the same mutation) or bi-allelic mutation withdifferent mutations on each allele are displayed. For colonies with twoindels, the number of times each allele was sequenced is displayed onthe right. In some cases, a third mutation or single wild-type allelewas sequenced, indicating that not all colonies are 100% clonal.Frame-shift alleles are indicated and mismatch nucleotides are denotedby lower-case text.

FIG. 9B: LDLR bi-allelic modification alleles, with notations as in FIG.9A.

FIG. 10: TALEN-induced deletions and inversions. A schematic of the DMDlocus is shown in panel (a). DNA orientation is denoted by blackchevrons. TALENs targeted to exons 6 and 7 (black arrowheads)co-transfected into male pig fibroblasts could result in a NHEJ fusionevent between exons 6 and 7. This could be identified using primers(black arrows) resulting in ˜500 bp amplicon. Panel b) SURVEYOR assay ofcells transfected simultaneously with TALENs targeted to exons 6 and 7reveal NHEJ indels at both sites. Percent NHEJ is displayed below. Panelc) PCR with primers flanking the presumptive deletion site yield a ˜500basepair product when both exon-6 and exon-7 TALENs are introducedsimultaneously, but not when transfected singly. Panel d) The predictedoutcome of an inversion event of the sequence between the TALEN targetsites is shown. DNA orientation is denoted by black chevrons. Primersoutside the presumptive flanking sites at the 5′ and 3′ end of theinversion locus are shown (black arrows) along with predicted productsize. PCR products were observed at both 5′ and 3′ junctions only whenboth exon-6 and exon-7 TALENs are introduced simultaneously.

FIG. 11: DMD deletion sequences. DMD deletion junctions from replicatetransfections are displayed. Above, exons 6 and 7 sequences are shaded,and TALEN-recognition sites are underlined. Inserted nucleotides areshaded.

FIG. 12: DMD inversion sequences. A schematic of the DMD inversionallele is shown with the 5′ and 3′ junctions (boxed) that were analyzedby sequencing. Below, the predicted sequence for each fusion is showncorresponding fusion at the center of each spacer for the TALEN pairs.TALEN-recognition sites are underlined. Sequenced inversion alleles froma transfected population are shown. The number of times each allele wassequenced is indicated at the right and inserted nucleotides areunderlined. Mismatched nucleotides are denoted as lower-case text.

FIG. 13: HDR induction in bovine fibroblasts. Panel a) TALENs(btGDF83.1, arrow) and a dsDNA template (BB-HDR) were designed tointroduce an 11-basepair deletion into exon-3 of bovine GDF8 (BelgiumBlue mutation) by Double-Strand Break-induced homologous recombination.Half of the binding site for the left TALEN is missing in the BB-HDRtemplate and thus should be resistant to TALEN cleavage. Panel b)SURVEYOR assay demonstrates activity of btGDF83.1 TALENs at both 37 and30° Celsius. The PCR product used for this assay was generated usingprimers b and b′ (shown in panel a). The BB-HDR template was notincluded in these replicates since it would confound estimates ofbtGDF83.1 activity. Panel c) Allele-specific PCR demonstrates that HDRinduction is dependent on co-transfection of TALENs and the BB-HDRtemplate. The PCR assay was developed to specifically detect HDRmodified GDF8 alleles using primers c and c′ (shown panel a). The 3′ endof primer c′ spans the 11-basepair deletion, and cannot amplify the wildtype allele (wt). Five hundred cell equivalents were included in eachPCR reaction including the positive control “C”. Percent HDR wasdetermined by comparative densitometry between experimental and controlreactions.

FIG. 14: Confirmation of Belgian Blue introgression by sequencing. Theschematics of Wagyu wild-type GDF8 (HDR Templates for GDF8 are SEQ IDNOs. 351-353) and the Belgian Blue template (BB-HDR) are shown. PCR wasconducted using primers located outside of the homology arms (c and d)on five PCR positive colonies followed by cloning and sequencing withprimer b′. Comparison to the wild-type sequence reveals the expected11-basepair deletion characteristic the Belgian Blue allele(heterozygous) in 4 of 5 colonies.

FIG. 15: Schematic and gel for TALEN-mediated HDR. A TALEN pair(LDLR2.1) targeted to the fourth exon of the swine low densitylipoprotein receptor (LDLR) gene was co-transfected with the supercoiledplasmid Ldlr-E4N-stop, which contains homology arms corresponding to theswine LDLR gene and a gene-trap enabling expression of Neomycinphosphotransferase upon HDR.

FIG. 16: Detailed sequence information for the Carlson +63 scaffold ofFIG. 5 and comparison to an alternative scaffold used by SangamoBiosciences.

FIG. 17: Detailed nucleic acid sequence for the vector used to make theCarlson +63 scaffold of FIG. 5, including non-translated portions.

FIG. 18: Use of an AAV-delivered single stranded DNA template emplatefor homologous recombination at the bovine GDF8 locus. a) TALENs(btGDF83.1, blue arrow) and a rAAV homologous recombination template(AAV-BB-HDR) were designed to introduce an 11 bp deletion into exon-3 ofthe bovine GDF8 gene (Belgium Blue mutation) by homologousrecombination. b) Allele-specific PCR demonstrates that HR induction isdependent on transfection btGDF83.1 TALENs and infection with defectiveAAV containing the AAV-BB-HDR template. The PCR assay was developed tospecifically detect HDR modified GDF8 alleles using primers c and c′(shown panel a). The 3′ end of primer c′ spans the 11 bp deletion, andcannot amplify the wild type allele “WT”. 1,000 cell equivalents wereincluded in each PCR reaction and positive control reactions with theindicated copy number of a control template were used for comparativequantification of homologous recombination.

FIG. 19: Use of single stranded oligonucleotides (ssOligos) as atemplate for homologous recombination at the bovine GDF8 locus. TALENs(btGDF83.1, arrow) and two ssODNs were designed to introduce an 11 bpdeletion into exon-3 of the bovine GDF8 gene (Belgium Blue mutation) byhomologous recombination. Each ssODN was 76 base pairs in length andwere sense and antisense strands of the same target site Allele-specificPCR demonstrates that HDR induction is dependent on transfectionbtGDF83.1 TALENs and subsequent transfection of ssODNs usingLipofectamine LTX 24 hours later. The PCR assay was developed tospecifically detect HDR modified GDF8 alleles using primers c and c′(shown panel a). The 3′ end of primer c′ spans the 11 bp deletion, andcannot amplify the wild type allele “WT”. 1,000 cell equivalents wereincluded in each PCR reaction and positive control reactions with theindicated copy number of a control template were used for comparativequantification of homologous recombination. BB-HDR Sense (S) has SEQ IDNO:133 and BB-HDR Anti (AS) has SEQ ID NO:134.

FIG. 20: Transfection of TALEN encoding mRNAs into livestock cellsresults in efficient target cleavage. Panel a: The indicated quantity ofmRNA was transfected into pig fibroblasts and transfected cells werecultured at either 30 or 37 degrees Celsius for three days prior toindel analysis. Panel b: Percent NHEJ was determined. The averagepercent NHEJ for by transfection of 4 micrograms of plasmid DNA encodingthe DMD7.1 TALENs is displayed by dashed lines for cells cultured at 30or 37 degrees Celsius.

FIG. 21: Transfection of mRNA encoded TALENs enhances ssODN HDR.btGDF83.1 TALEN mRNA and BB-HDR sense ssODN (SEQ ID NO:133) wereintroduced into Wagyu cells by the specified mechanism and HDR wasmeasured by PCR assay described above. Colonies were isolated from thepopulation of cells where both TALEN mRNA and the ssODNs were deliveredsimultaneously by nucleofection.

FIG. 22: Introgression of naturally occurring alleles within a speciesusing mRNA encoded TALENs and ssODNs. The Piedmontese Myostatin alleleC313Y was introgressed into Waygu fibroblasts by the methods of FIG. 21using btGF83.6-G (SEQ ID NO: 351). The sequence labeled “oligo” is hasSEQ ID NO:503.

FIG. 23: The process of FIG. 22 was repeated at a different temperature(37° C.) using btGF83.6-G (SEQ ID NO: 351).

FIG. 24: Introgression of naturally occurring alleles from one speciesto another using mRNA encoded TALENs and ssODNs. The PiedmonteseMyostatin allele C313Y was introgressed into Ossabaw fibroblasts by themethods of FIG. 25. The following ssODN was usedggccaattactgctctggagagtatgaattcgtatttttacaaaaataccctcacactcatcttg (SEQID NO:146)

FIG. 25: Introduction of a particular frameshift allele into porcineLDLR using mRNA encoded TALENs and ssODNs. A 90-bp oligo was created tointroduce a 4 base pair insertion into exon 2 of the porcine LDLR gene.The insertion creates a novel BamH1 site and is predicted to cause aframeshift allele. After co-transfection of ssLDLR2.1 TALEN mRNA (atindicated dosage in micrograms) and 0.3 nMol of ssODN cells werecultured at 30.0 for 3 days, followed by an additional day at 37° C.NHEJ was measured by SURVEYOR assay at days 4 and 20. Percent HDR wasdetermined by BamH1 digest of PCR products that include exon 2 ofporcine LDLR and quantification of restriction fragments (indicative ofHDR) and comparison to wild type products (top product, no HDR) bydensitometry. Colonies were isolated from each treatment and analyzed byPCR and BamH1 digest. The sequence labeled “Wt” has SEQ ID NO:523 andthe sequence labeled “Sense” has SEQ ID NO:524.

FIG. 26: DNA and mRNA encoded TALENs are active in stem cells. The toppanel shows percent NHEJ of DMD7.1 TALENs transfected as plasmid DNAinto porcine male germ-line stem cells (GSCs). Nucleofection solutionsL, V or B were evaluated. The lower panel shows SURVEYOR assay resultsof porcine GSCs transfected with mRNA encoding DMD7.1 TALENs. Thequantity of mRNA (micrograms) is indicated.

FIG. 27: TALENs mediate DSB in chicken cells and can stimulatehomologous recombination in chicken primordial germ cells (PGCs). Panela) TALEN activity was first determined in DF1 immortalized chicken cellsline transfection and SURVEYOR assay. Panel b) Schematic depiction ofthe targeting strategy of the chicken ddx4 locus. The GFP/Puromycinreporter gene will replace the endogenous coding sequence in the secondexon of targeted cells. Penal c) PGCs were transfected with thehomologous recombination construct, TALENs (either Tal 1.1 or Tal7.1,empty vector) and a puromycin selection transposon. After selection inpuromycin, GFP+ cells could be isolated when Tal 1.1 TALENs were used(right picture, left is bright field) but not with Tal7.1 TALENs orempty vector transfections.

FIG. 28: Schematic and gel for the TALEN-mediated HDR of FIG. 27.

FIG. 29: Transgenic swine created by the processes of FIGS. 27 and 28.

FIG. 30: Illustrates a general process of using a TAL-effectorendonuclease (TALEN).

FIG. 31: Illustrates a general process of using a Cas9/CRISPRendonuclease (an RNA-guided endonuclease).

FIG. 32: Illustrates the theory of operation for TALENs that explainswhy they are generally ineffective for making SNP changes; similarprocesses apply to other targeted endonucleases.

FIG. 33: Illustrates a general method of making and using targetingendonucleases that is effective to make an SNP edit.

FIG. 34: Illustrates another general method of making and usingtargeting endonucleases that is effective to make an SNP edit.

FIG. 35: TALEN-mediated introgression of POLLED. Panel a) A schematic ofthe strategy to introgress the Polled allele into Holstein (HORNED)cells. The POLLED allele, bottom, is a tandem repeat of 212 bp(horizontal arrow) with a 10 bp deletion (not shown). TALENs weredeveloped to specifically target the HORNED allele (vertical arrow)which could be repaired by homologous recombination using the POLLED HDRplasmid. Panel b) Representative images of colonies with homozygous orheterozygous introgression of POLLED. Three primer sets were used forpositive classification of candidate colonies: F1+R1, F2+R2 and F1+P(POLLED specific). Identity of the PCR products was confirmed bysequencing F1+R1 amplicons.

FIG. 36: A plot of experimental data generated for evaluation oftransfected mRNA as a source of TALENs. TALENs were introduced intofibroblasts encoded by either unmodified mRNA, modified mRNA (mod mRNA)or plasmid DNA (pDNA). Two quantities of each TALEN preparation weretransfected into cells subsequently cultured 3 days at 30° C. or 37° C.prior to analysis of indels, reported as % NHEJ.

FIG. 37A: A plot showing that an mRNA source of TALENs stimulatedefficient and consistent HDR using an oligo donor. Each chart displaysresults of targeting a specific locus in fibroblasts (e.g., ssIL2RG;“ss” for Sus scrofa and “bt” for Bos taurus) using oligo donor templatesand TALENs delivered as plasmid DNA or mRNA. (Insets) Diagrams of theoligo templates, in which the shaded boxes represent the TALEN-bindingsite and the spacers are shown in white. Each oligo contains either a4-bp insertion (ins4) or deletion (del4) that introduces a novelrestriction site for RFLP analysis. Presumptive BMs replace theconserved −1 thymidine (relative to the TALEN-binding site) with theindicated nucleotide. Fibroblasts were transfected with eitherTALEN-encoding plasmids (3 μg) or mRNA (1 μg) along with 3 μM of theircognate oligo-homologous template. Cells were then incubated at 37° C.or 30° C. for 3 d before expansion at 37° C. until day 10. TALENactivity was measured by the Surveyor assay at day 3 (Day3 Surveyor),and HDR was measured at days 3 and 10 by RFLP analysis (Day3% HDR andDay10% HDR). Each bar displays the average and SEM from threereplicates.

FIG. 37B: A plot of experimental data generated to evaluate kinetics ofTALEN induced HDR with oligonucleotide templates. Cells were transfectedwith either TALEN-encoding mRNA or plasmid DNA and oligos with 4 basepair insertions targeting LDLR or APC genes. Panel a) RFLP analysis oncell populations at indicated time points. Panel b) Results from panela, were quantified by densitometry and the averages were plotted as afunction of time with SEM (n=3). HDR signal first appears 12 hourspost-transfection and accumulates over time.

FIG. 38: A plot of experimental data generated to evaluate influence ofmutation type on the frequency of HDR. Panel a) The wildtype ssLDR (SEQID NO:241) and sequence of five oligos used to target ssLDLR: (from topto bottom: SEQ ID NOS: 242, 243, 244, 245, and 246). TALEN binding sitesare indicated in boxed text and the novel BamHI site is underlined. SNPsincluding BMs and insertions are circled. Panel b) Cells weretransfected with LDLR2.1 TALEN mRNA (1 μg) and oligos (2 μM final). HDRat day 3 was determined by RFLP analysis and the average with SEM (n=3)was plotted. Panel c) Cattle cells were transfected with btRosa1.2 TALENmRNA and either 41 mloxP or 60 loxP oligos (2 μM final). The numbers 41and 60 refer to the number of homologous bases. Each oligo contains a 34bp loxP site, either a modified (mloxP) or wild type (loxP) version, inthe center of the spacer.

FIG. 39: CRISPR/Cas9 mediated HDR to introgress the p65 S531P mutationfrom warthogs into conventional swine. Panel a) The S531P missensemutation is caused by a T-C transition at nucleotide 1591 of porcinep65. The S-P HDR template includes the causative TC transition mutation(oversized text) which introduces a novel XmaI site and enables RFLPscreening. Panel b) Cells were transfected with S—P-HDR oligos (2 μM),two quantities of plasmid encoding hCas9 (0.5 μg or 2.0 μg); and fivequantities of the G2A transcription plasmid (0.05 to 1.0 μg). Cells fromeach transfection were split 60:40 for culture at 30 and 37° C.respectively for 3 days before prolonged culture at 37° C. until day 10.Surveyor assay revealed activity ranging from 16-30%. Panels c and d)RFLP analysis of cells sampled at days 3 and 10. Expected cleavageproducts of 191 and 118 bp are indicated by black arrows. The two gRNAsequences are P65_G1S (SEQ ID NO:247) and P65_G2A (SEQ ID NO:248). Thewild type porcine p65 is SEQ ID NO:249, shown in alignment with thehomology directed repair (HDR) template S—P-HDR (SEQ ID NO:250). Theleft TALEN sequence and right TALEN sequence to bind p65 DNA are SEQ IDNOs: 251 and 252, respectively.

FIG. 40: Experimental data for comparison of TALENs and CRISPR/Cas9mediated HDR. Panel a) APC14.2 TALENs (SEQ ID NOS:253 and 254) and thegRNA sequence APC14.2 G1a (SEQ ID NO:255) are shown relative to the wildtype APC sequence (SEQ ID NO:256). Below, the HDR oligo (SEQ ID NO:257)is shown which delivers a 4 bp insertion (boxed text) resulting in anovel HindIII site. Cells were transfected with HDR template, and TALENmRNA, plasmid DNA encoding hCas9 and the gRNA expression plasmid; ormRNA encoding hCas9 plus the gRNA expression plasmid, cultured at either30 or 37° C. for 3 days before expansion at 37° C. until day 10. Panelb) Charts displaying RFLP and Surveyor assay results

FIG. 41: Experimental data for SNP introgression using oligo donors.Panel a) is a plot of maintenance of HDR alleles with or withoutblocking mutations (BMs) for pig LDLR and GDF8. Each oligo had the sameSNPs/restriction 313 site plus or minus BMs. Average homologousrecombination and SEM (n=3) is shown. Panel b) shows results forintrogression of myostatin C313Y into Wagyu fibroblasts. The C313Ymissense mutation is caused by a G-A transition (indicated by oversizedtext) at nucleotide 938 of bovine myostatin. The HDR template (labeleddonor, SEQ ID NO:258), also includes a T to C transition (circled) tointroduce a novel EcoRI site for RFLP screening. Two left TALENs weredesigned against the locus, btGDF83.6-G (SEQ ID NO:259), targeting thewild type alelle (Wt) (SEQ ID NO:260), and btGDF83.6-A (SEQ ID NO:261),targeting the mutant allele (C313Y); both share a common right TALEN(SEQ ID NO:262). Transfection, culture and measurement were conducted asabove. The average and SEM for btGDF83.6-G (n=30) and btGDF83.6-A (n=5)represent twelve and three biological replicates, respectively. Atwo-sided student's t-test was used to compare averages between groups;the p values are indicated.

FIG. 42: A plot that shows results for sequence analysis of TALENstimulated HDR alleles. The count of perfect, intended HR reads versusthe wild type reads is plotted for insertion (panel a) and SNP alleles(panel b). The target locus, time point and whether or not BMs wereincluded in the oligo are indicated. Panel c). Reads from btGDF8 and p65sorted for incorporation of the target SNP and classified as intended(iSNP) versus those with an additional mutation (iSNP+Mut) and plottedagainst the total number of reads.

FIG. 43: Results of sequence analysis of HDR alleles. Sequencing readscontaining the correct insertion (Panel a) or SNP allele (Panel b) wereanalyzed for incorporation of BM. The target locus, time point andwhether or not BMs were included in the oligo are indicated below eachgraph. Panel c). The data of FIG. 13 panel c was further classified bymutation type and compared. Some reads contained only the iSNP, othershad a concomitant indel (iSNP+indel), or a concomitant unintended SNP(iSNP+uSNP).

FIG. 44: Experimental data for multiple SNPs placed in the TALENDNA-binding site to stabilize HDR alleles in the EIF4GI gene. Panel a)shows a portion of wild type EIF4GI Wt-NL (SEQ ID NO:263) and a pair ofTALENs (SEQ ID NOS: 264 and 265) designed to cut the wild type EIF4GI tostimulate homologous recombination. Also aligned to the Wt sequence isthe core sequence (SEQ ID NO:266) of the donor oligo, DF-HDR, used tointroduce three SNPs (underlined oversized letters) into the genome. Thethird SNP creates a novel EagI restriction site that was used for RFLPanalysis. Pig fibroblasts were transfected with EIF4GI14.1 TALEN mRNA (2μg) and DF-HDR (2 μM) and then cultured at 30° C. for 3 days prior toanalysis and colony propagation. Panel b) shows RFLP analysis onpopulation three days post transfection. Expected product sizes of 344,177 and 167 bp are indicated by filled triangles. Panel c) shows RFLPassay on isolated cellular clones. Day 3 cells were used to derivemonoclonal colonies through dilution cloning. An example of colonieswith heterozygous (open triangles) or homozygous (filled triangles) HDRalleles are indicated.

FIG. 45: A plot of data for hypothermic treatment maintenance of SNP HDRalleles. Pig fibroblasts were transfected with TALEN mRNA (1 μg) andoligos (3 μM). Cells from two independent transfections were pooled foreach replicate and evenly distributed into six wells of a 6-well plateand cultured at 30° C. Samples were collected from these populations forRFLP analysis on days 1-7 (minus day 6, 1D to 7D along X-axis)post-transfection and the remaining cells were transferred to 37° C.Samples for each condition were collected again at day 12 for RFLPanalysis. The average HDR and SEM (n=3) is shown at the initialcollection and once again at day 12.

FIG. 46: Experimental results for TALENs made with intentional RVDmismatches to improve frequency of correct alleles when introducing aSNP. Panel a) shows a TALEN pair (caCLPG 1.1, SEQ ID NOs: 267-270, topto bottom, left to right) designed to target the caCLPG region. Oligodriven HDR was utilized to introduce the desired Adenine to Guanine SNP(the targeted Adenine is boxed). The desired SNP allowed genotyping by aloss of an AvaII restriction site. Each TALEN monomer is indicated inshading above their respective binding locations. Panel b) A caCLPGwildtype sequence is shown (SEQ ID NO:271). Each allele of single-cellderived colonies that were resistant to AvaII were sequenced (fourteensequences with SEQ ID NOS: 272-277, 279, 278 and 280-285, from top tobottom). All of the alleles that contained the SNP of interest (boxed)also contained deletions (marked with dashes in the AvaII ResistantAllele sequences) or insertions (marked with dashes in the WT sequence).In panel c), intentional mismatches (italicized circled text) wereintroduced into the RVD sequence. The desired SNP (boxed) was in theright monomer of the TALEN. Panel d) shows TALEN activity as measuredvia a Cell assay. The percent of non-homologous end joining (% NHEJ) isindicated for each was measured. Panel e) shows both an alignment of acaCLPG wildtype sequence is shown (SEQ ID NO:294) with sequenced allelesof AvaII-resistant single-cell derived colonies produced with caCLPG1.1c (six sequences, with SEQ ID NOS: 295, 298 and 297-300, top tobottom). The desired SNP is boxed. Colony 37 and 78 were heterozygousfor the desired SNP and showed no additional indels. Colony 142 washomozygous for the desired SNP, but contained a 4 bp insertion on oneallele.

FIG. 47: Results for experiments to introgress a SNP with and without amismatch in the targeting endonuclease. Panel a) shows a schematic ofthe bovine DGAT sequence around K323A (SEQ ID NOs: 301 and 302). Thegrey arrows represent the TALEN monomers where they bind to the DGATsequence. The left arm consists of 16 RVDs, the right arm consists of 15RVDs, and the spacer is 16 base pairs long. The GC and gga gct, boxed,are the targeted base pairs. The DGAT oligo converts the GC to an AA tocreate the desired DGAT mutant. As a marker for HDR, the boxed GGGAGC isconverted to AAGCTT that creates a novel HindIII restriction site. Sincethis change is in the spacer, it should not affect TALEN binding as tonot interfere with the intentional mismatch results. Panel b) DGAT TALENRVD sequences. btDGAT 14.2 contains no intentional mismatches in theRVDs. btDGAT 14.4, 14.5, and 14.6 each contain one intentional RVDmismatch at either position 1, 3, or 5 of the left TALEN monomer(circled). Panel c) Bovine fibroblasts were transfected with 1 ug oftalen and 0.4 nmoles of oligo. Three days after transfection cells werelysed, the DGAT sequence was amplified by PCR, digested with HindIII andran on an acrylamide gel. The percent efficiency of HDR was determinedby densitometry (HR). Panel d) Sequence analysis of colonies producewith the original 14.2 TALENs. Of twelve colonies, none that werepositive for the HindIII RFLP contained the desired mutation due toindels overlapping the site. (From top to bottom, SEQ ID NOs: 311 to319, 321). Panel e) Colonies derived from TALENs 14.5 and 14.6 producedthe correct DGAT mutation and HindIII restriction site. These two TALENpairs produced a total of two homozygous (HH) and three heterozygous(Hh) colonies. TALEN 14.4 did not produce any colonies with the correctDGAT mutation (data not shown), from top to bottom, SEQ ID NOs: 322 to327.

FIG. 48: Sets forth the process of TALEN-HDR/RMCE. The foxed cassette istransfected along with TALENs compatible with the oligo, the loxP oligoand a source of Cre recombinase. The bar graph shows the number ofpuromycin resistant colonies produced by this method when YFC-Cre versusmCherry was included in the transfection. To confirm targeting to theSRY locus, PCR was conducted across the predicted junction (shown) willresult in a 370 bp product. This product is apparent only when Cre isincluded.

FIG. 49: An illustration of a process of making and using animalsgenetically modified for control of maturation.

FIG. 50: Confirmation of Belgian Blue introgression by sequencing. Theschematics of Wagyu wild-type GDF8 and the Belgian Blue template(BB-HDR) are shown. PCR was conducted using primers located outside ofthe homology arms (c and d) on five PCR positive colonies followed bycloning and sequencing with primer b′. Comparison to the wild-typesequence revealed the expected 11-basepair deletion characteristic theBelgian Blue allele (heterozygous) in 4 of 5 colonies.

FIG. 51: Introgression of naturally occurring alleles from one speciesto another using mRNA encoded TALENs and ssODNs. The PiedmonteseMyostatin allele C313Y was introgressed into Ossabaw.

FIG. 52: Modification of targeted genes. Each chart displays results oftargeting a specific locus in fibroblasts (e.g., ssIL2RG; “ss” for Susscrofa and “bt” for Bos taurus). (Insets) Diagrams of the oligotemplates, in which the shaded boxes represent the TALEN-binding siteand the spacers are shown in white. HDR was measured at days 3 and 10 byRFLP analysis (Day3% HDR and Day10% HDR). Each bar displays the averageand SEM from three replicates.

FIG. 53: Sequence analysis of TALEN stimulated HDR alleles. PCRamplicons flanking the target site (200-250 bp total) derived from TALENmRNA and oligo transfected cell populations were sequenced by ILLUMINAsequencing. Total read count ranged from 10,000 to 400,000 per sample.The count of perfect, intended HR reads versus the wild type reads isplotted for insertion (panel a) and SNP alleles (panel b). The targetlocus, time point and whether or not BMs were included in the oligo areindicated below. Panel c). Reads from btGDF8 and p65 were sorted forincorporation of the target SNP and then classified intended (iSNP)versus those with an additional mutation (iSNP+Mut) and plotted againstthe total number of reads.

FIG. 54: Cloned pigs with HDR alleles of DAZL and APC. (A) RFLP analysisof cloned piglets derived from DAZL- and APC-modified landrace andOssabaw fibroblasts, respectively. Expected RFLP products for DAZLfounders are 312, 242, and 70 bp (open triangles), and those for APC are310, 221, and 89 bp (filled triangles). The difference in size of the312-bp band between WT and DAZL founders reflects the expected deletionalleles. (B) Sequence analysis confirming the presence of the HDR allelein three of eight DAZL founders, and in six of six APC founders. BMs inthe donor templates (HDR) are indicated with arrows, and inserted basesare enclosed in blocks. The bold text in the top WT sequence indicatesthe TALEN-binding sites. FIG. 54 panel (B) discloses SEQ ID NOS 182-195,respectively, in order of appearance.

FIG. 55: A schematic of porcine GPR54 and the gene targeting strategyfor knockout is depicted in panel a. TALENs designed to bind exon 3(underlined text) were co-transfected with an oligonucleotide homologytemplate (HDR) designed to introduce a premature stop codon and aHindIII restriction site. FIG. 55 panel a discloses SEQ ID NOS 196 and519, respectively, in order of appearance. Panel b: 2 micrograms ofTALENs encoding mRNA plus 0.2 nMol of the HDR template were transfectedinto pig fibroblasts that were grown as colonies and analyzed forhomology dependent repair by HindIII RFLP assay. PCR results are shown;each lane represents one colony. Cleavage products of 231 and 158 bp areindicative of homology dependent repair. Colonies with the parent bandof 389 bp are classified as heterozygous (open triangle) and thosewithout are classified as homozygous (filled triangle) for the HDR,knockout allele.

FIG. 56: Panel a: Nucleotide and deduced translated amino acid sequenceof mRNA encoding tilapia kisspeptin. The structural organization of thekiss gene is conserved and contains two coding exons, one encoding boththe signal peptide and part of the kisspeptin precursor, the otherencoding the remainder of the precursor including the kisspeptin-10sequence. The position of the intron is indicated by a triangle glyph.The location of the forward and reverse primers for PCR amplification ofthe target region (442 bp) are shown. The binding sites for the twoengineered pairs of TALENs, Kiss1.1a and Kiss1.1b are indicated in blackand gray boxes. FIG. 56 panel a discloses SEQ ID NOS 198 and 564-565,respectively, in order of appearance. Panel b shows a schematicrepresentation of the targeted kiss genomic region showing the locationof the kisspeptin-10 biologically active peptide and each kiss1.1a and1b TALENs recognition sites. PCR (442 bp) and qPCR primer pairs (138 bpamplicon) used for analysis of indels are shown as well.

FIG. 57: Panel a: Nucleotide and deduced translated amino acid sequenceof mRNA encoding tilapia GPR-24 mRNA. The structural organization of thekissr gene is conserved and contains five coding exons. The positions ofall four introns are indicated by a triangle glyph. The KissRE2 andKissRE3 TALENs targeted loci are located in the coding exon 2 (whiteboxes) and 3 (gray boxes) respectively. The location of the sense Leftand antisense Right TALENs recognition sites are shown in boxes. FIG. 57panel a discloses SEQ ID NOS 528-529 and 566, respectively, in order ofappearance. Panel b shows a schematic representation of the tilapiaGPR-24 genomic region showing the location of the introns (Strokedgoalpost), the coding exons 2 and 3 (black arrows) containing thekissRE2 and RE3 loci (white boxes). The location primers used for PCRand qPCR analysis and the size of the corresponding amplicons are shownas well.

FIG. 58: Melt analysis of 100-120 bp qPCR product containing the kissand kissRE3 loci. Panels a and b show melting curves of ampliconsgenerated from the gDNA extracted from the fin of fish treated kiss1.1aand kissRE3 TALENs pairs. The plain arrows point to melting profiles(panel a) or (panel b) that were significantly different than thoseobtained from untreated fish (dotted arrows) and correspond to candidatemutant fish kiss #41, RE3 #1, 4, 6 and 11. Panel c: A 442 bp genomicsegment containing the targeted Kiss loci was PCR amplified from—TALENtreated fish #41. The PCR product was cloned into TOPO 2.1 TA vector,and transformant colonies were hand-picked for direct QPCR analysis. Theplain arrows point to selected melt profiles obtained from coloniescontaining different deletions at the kiss loci. Panel d: To bettervisualized the varied mutations cloned, we graphed our QPCR colonyscreen on a scatter plot of Cts versus melt temperature, where eachclone is represented by a data plot (x, y) with x representing its Ctand y representing its melt temperature. The graph represent coloniescontaining the 702 bp PCR fragment amplified from Fish RE3 #4. Melttemperature below that of a wild type sequence all contained the kissRE3amplicon with varied deletions at the target site. Cts: Cyclethresholds.

FIG. 59: Description of somatic mutations induced by engineered TALENsat the kiss gene (site kiss1.1a) (panel a) and kissR gene site (KissRE3)(panel b). The wild-type sequences are shown at the top of each panelwith the sense left and antisense right TALEN recognition element sitesshown in bold highlighted in dark gray and the sense spacer highlightedas underlined text. Deletions are shown as dashes and insertions aslower case letters highlighted in light gray. The net change in lengthcaused by each indel mutation is to the right of each sequence (+,insertion; −, deletion). A few alterations have both a deletion and aninsertion of sequence. The number of times each mutant allele wasisolated is shown in brackets. FIG. 59 panel a discloses SEQ ID NOS 202,530-532 and 206-211 and FIG. 59 panel b discloses SEQ ID NOS 212, 533,214-216 and 534-535, all respectively, in order of appearance.

FIG. 60: Panel a: Selected sequencing chromatography of PCR productsfrom two sibling progeny in line KissRE3 #11. These graphs indicate thepresence of mutation reading simultaneously a kissRE3 mutant and a WTallele. Boxes indicate matching nucleotides on the mutant and WT allelesand arrow points to the location where sequences become divergent andthus where these deletion begin. To characterize the mutation weanalyzed the pattern of unique nucleotide reads in the sequence (wherethe chromatograph show above background non duplicate nucleotide reads).By shifting the WT sequence and increased size deletion sequences, wefound that a 7 pb and 5 bp deletions reproduce the pattern of singlenucleotide reads on these chromatograph. FIG. 60 panel a discloses SEQID NOS 219, 222, 220, 536 and 537, respectively, in order of appearance.Panel b: Description of all inherited indel mutations induced byengineered TALENs at the kiss gene (kiss1.1a site, top) and kissr gene(KissRE3 site, bottom). The wild-type sequences are shown at the topwith the sense left and antisense right TALEN recognition elements shownin bold letter highlighted in dark gray and the sense spacer highlightedas underlined text. Deletions are shown as dashes. The net change inlength caused by each indel mutation is to the right of each sequence(−, deletion). The number of times each mutant allele was isolated isshown in brackets. FIG. 60 panel b discloses SEQ ID NOS 202, 208, 226,212, 228, 214, 230-232 and 538, respectively, in order of appearance.Panel c: Description of the most severe lesions found at the kiss andkissRE3 sites. The 18 nt deletion at the kiss1.1a loci result in theloss of 6AA (underlined) 3 of which are from the core sequence of thekisspeptin-10 active peptide (highlighted in gray). The 7 nt deletion atthe kissRE3 loci (underlined text) result in significant alteration ofthe gene product with two AA substitution immediately followed by a stopcodon. The resulting protein is C-terminally truncated by 215 AA. FIG.60 panel c discloses SEQ ID NOS 234-237, 539 and 239, respectively, inorder of appearance.

FIG. 61: Panel a) Schematic of the bovine horned/polled locus. TALENswere designed to cut the horned variant where indicated by arrowheads.Panel b) The sense strand sequence of four TALENs. Panel c) Surveyorassay of horned Holstein fibroblasts cells three days post transfectionwith mRNA encoding each TALEN pair. TALEN ID and incubation temperaturepost transfection are indicated above the gel. Sequence identifiers asfollows: HP1.1 left and right (SEQ ID NOs: 240 and 347); HP1.2 left andright (SEQ ID NOS: 348 and 149); HP1.3 left and right (SEQ ID NOS: 150and 151); HP1.4 left and right (SEQ ID NOS: 152 and 153).

FIG. 62: TALEN-mediated introgression of POLLED. Panel A) A schematic ofthe strategy to introgress the Polled allele into Holstein (HORNED)cells. The POLLED allele, bottom, is a tandem repeat of 212 bp (redarrow) with a 10 bp deletion (not shown). TALENs were developed tospecifically target the HORNED allele (green vertical arrow) which couldbe repaired by homologous recombination using the POLLED HDR plasmid.Panel B) Representative images of colonies with homozygous orheterozygous introgression of POLLED. Three primer sets were used forpositive classification of candidate colonies: F1+R1, F2+R2 and F1+P(POLLED specific). Identity of the PCR products was confirmed bysequencing F1+R1 amplicons.

FIG. 63: Example of polled conversion in an isolated colony. Individualcolonies were propagated from cell populations described in FIG. 2. Eachcolony was analyzed by the PCR method described in FIG. 2. Clone 3 has aproduct at both 389 and 591 bp (arrow) indicative of a heterozygousconversion to the polled allele. The Repair Template used was 591residues in length.

FIG. 64: Panel a) Schematic to convert a horned allele to a polledallele. HP1.3 TALENs plus a short repair template are introduced intohorned cells. The repair template was generated by PCR from polled Angusgenomic DNA; homology lengths are indicated. Panel b) PCR assessment ofpolled conversion in horned Holstein fibroblasts transfected with 2 μgof TALEN mRNA+500 ng of ssDNA coated with Gal4:RecA. Each lane/PCRreaction consists of ˜3 cell equivalents diluted from a transfectedpopulation. PCR using primers btHP-F1 and btHP-R1 from horned cellsresults in a product of 389 bp. Conversion to polled results in a netinsertion of 202 base pairs; thus the PCR product of the same primersresults in a 591 bp product (arrow in left margin). The number ofreactions with products indicative of polled conversion is shown in theupper right corner. Panel c) PCR assessment of polled conversion inhorned Holstein fibroblasts transfected with 2 ug of TALEN mRNA+1,500 ngof ssDNA. The number of reactions with products indicative of polledconversion is shown in the upper right corner.

FIG. 65: Comparison of TALENs and CRISPR/Cas9 mediated HDR at porcineAPC. Panel a) APC14.2 TALENs (SEQ ID NOS: 154 and 155) and the gRNAsequence APC14.2 G1a (SEQ ID NO: 157) are shown relative to the wildtype APC sequence (SEQ ID NO: 156). Below, the HDR oligo (SEQ ID NO:158) is shown which delivers a 4 bp insertion resulting in a novelHindIII site. Pig fibroblasts transfected with 2 μM of oligo HDRtemplate, and either 1 μg TALEN mRNA, 1 μg each plasmid DNA encodinghCas9 and the gRNA expression plasmid; or 1 μg mRNA encoding hCas9 and0.5 m of gRNA expression plasmid, were then split and cultured at either30 or 37° C. for 3 days before expansion at 37° C. until day 10. Panelb) Charts displaying RFLP and Surveyor assay results.

FIG. 66: Schematic of the bovine PRLR gene (SEQ ID NO: 525) and positionwithin the genome. A mutation that leads to truncation of the PRLR geneis created, which corresponds to the SLICK 1 allele. The CG→G mutationcauses a frame shift and termination at amino acid 461 (Trunc461). TheXbaI site was included in one of the HDR template designs to enable RFLP(SEQ ID NOS: 383 and 384).

FIG. 67: Schematic of the bovine PRLR gene (SEQ ID NO: 525). Holsteincells were transfected with 1 μg PRLR9.1 TALENs along with 0.4 nmol ofthe HDR template, PRLR9.1 (SLICK1) XbaI RE site (SEQ ID NO: 383). Threedays after transfection, the population of cells was screened for TALENactivity by Cell and RFLP assay (left gel). Remaining cells werere-plated for derivation of individual colonies and screened by RFLPassay (right gel). Many candidates were identified, and sequenceanalysis revealed that clones 11 and 93 were positive for preciseintrogression of SLICK1.

FIG. 68: Pictures of live POLLED Holstein calves.

FIG. 69: Pictures of a POLLED Holstein calf eye showing an extendedeyelash length indicative of a homozygous POLLED phenotype.

FIG. 70: Comparison of the eyelashes of a horned bull (A) as compared toPOLLED cattle (B-E) with the same allele introgressed into the POLLEDHolstein seen in FIGS. 68 and 69 (Allais-Bonnet et al., (2013) NovelInsights into the Bovine POLLED Phenotype and Horn Ontogenesis inBovidae. PLoS ONE 8(5): e63512.doi:10.1371/journal.pone.0063512).

FIG. 71: Picture of an Angus/Holstein crossbreed (see the World Wide Web(www) at albertachickensetc.punbb-hosting.com/viewtopic.php?id=15272).

FIG. 72: Chromosome 1: Celtic POLLED allele homology template. 202 bpPOLLED allele present in Celtic Allele double underlined, 212 bp Hornedallele (SEQ. ID NO: 526) includes underlined and double underlinedsequence. The double underlined region is deleted to create POLLEDallele. The 5′ homology arm (before the double underlined sequence) isindicated by the box, 3′ homology arm (after the double underlinedsequence) is indicated by the box.

FIG. 73: CRISPR/Cas9 target sequence, ssKiss1 Exon 2 and the repairtemplate ssKiss1 Ex2.9 for porcine fibroblast Kiss knockout experiment.FIG. 73 discloses SEQ ID NOS 540-541, 394 and 542-547, respectively inorder of appearance.

FIG. 74: CRISPR/Cas9 target sequence, ssKiss1 Exon 2, and the repairtemplate ssKiss1c.2.9 Blocking HDR designed to be inserted by HDR withinssKiss1 Exon 2 for porcine fibroblast Kiss knockout experiment. FIG. 74discloses SEQ ID NOS 540-541 and 548-553, respectively, in order ofappearance.

FIG. 75: Efficiency of blocking HDR oligo in fibroblast population. Theblocking oligo changes the CGG PAM to CAG and introduces a silentmutation to generate an AcuI restriction site. The gel shows the resultsof RFLP analysis, indicating the inclusion of the AcuI restriction site.FIG. 75 discloses SEQ ID NOS 540-541 and 554-557, respectively, in orderof appearance.

FIG. 76: Kiss swine fibroblast transfection population data without theBlocking HDR oligo. Panel A shows the RFLP analysis of male pigfibroblasts next to transfected (+) or non-transfected (C) controls.Cells were transfected with a combination of IDT Alt-R crRNA:Tracer RNAcomplex, Cas9=Alt-R HiFi Cas9 nuclease (protein) and ssKiss1 c.2.9 HD3HDR. Results show RFLP analysis 3 days after transfection. Panel B showsRFLP analysis of Female pig fibroblasts transfected with IDT Alt-RcrRNA:Tracer RNA complex, Cas9=Alt-R HiFi Cas9 nuclease (protein) withand without the ssKiss1 c.2.9 HD3 HDR template. Results show RFLPanalysis 3 days after transfection. Cells from these populations wereplated at low density for isolation of single cell derived colonies andevaluated for editing. Select homozygous HDR clones were confirmed bySanger Sequencing and used for cloning founder animals.

FIG. 77: Kiss Colony RFLP Image. Individual colonies were propagatedfrom the transfected population above and subjected to RFLP analysis foridentification of mutant colonies. Three outcomes were apparent: (1)Wild Type (WT) RFLP result, (2) Mutant RFLP result (this population wassequenced and used in bi-allelic mutant animals), (3) Heterozygous RFLPresult. The letter A indicates another example mutant result which wasnot used in cloning.

FIG. 78: Confirmation of HDR in RFLP positive colonies by SangerSequencing. Row 1 shows the consensus sequence from the alignment. Row 2shows the predicted sequence from Ensembl. Row 3 shows sequence ofuntransfected kiss1 cells. Rows 4-6 show sequence of from coloniesisolated after transfection with the gene editing reagents. These areexamples of colonies with homozygous HDR with the ssKiss1 c.2.9 HD3 HDRas indicated by the 8 bp insertion. The colony represented in row 5 wasused for production of founder animals by cloning. FIG. 78 discloses SEQID NOS 393, 389 and 558-561, respectively, in order of appearance.

FIG. 79: Confirmation of Kiss1 knockout by HDR in pig zygotes. 25 ng/μlgRNA; 50 ng/μ1 Cas9; 33.3 ng/μ1 HD3 HDR; 66.7 ng/μ1 Blocking HDRMicroinjection: n=18. The injected zygotes were cultured to blastocyststage prior to whole genome amplification, PCR over the target site, andSanger Sequencing. Amplicons were sequenced using Sanger Sequencingfollowed by analysis using ICE software (Synthego). Results indicatethat about 22% of injected zygotes were heterozygous (31-60% mutant).Guide RNA is IDT Alt-R crRNA: Tracer RNA complex, Cas9=Alt-R HiFi Cas9nuclease (protein). HDR templates are IDT ssDNA oligonucleotides.

FIG. 80: Confirmation of Kiss1 knockout by HDR in pig zygotes. 25 ng/μ1gRNA; 25 ng/μ1 Cas9; 26.7 ng/μ1 HD3 HDR; 53.3 ng/μ1 Blocking HDRMicroinjection: n=11. The injected zygotes were cultured to blastocyststage prior to whole genome amplification, PCR over the target site, andSanger Sequencing. Amplicons were sequenced using Sanger Sequencingfollowed by analysis using ICE software (Synthego). Results indicatethat about 9% of injected zygotes were heterozygous (31-60% mutant).Guide RNA is IDT Alt-R crRNA: Tracer RNA complex, Cas9=Alt-R HiFi Cas9nuclease (protein). HDR templates are IDT ssDNA oligonucleotides.

FIG. 81: Development of a gene edit cell line of GPR54 knock out pigfibroblasts. A PCR-RFLP assay was run to confirm that a frame shift andpremature stop codon were successfully integrated into Exon 3 of theGPR54 gene using TALENS. FIG. 81 discloses SEQ ID NOS 196 and 562-563,respectively, in order of appearance.

FIG. 82: Confirmation of the GPR54 knockout from FIG. 81 in pig embryosproduced by somatic cell transfer and grown to piglets afterimpregnanton within sows.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed precise, high frequency editing of avariety of genes in various cells and/or animals that are useful foragriculture, for research tools, or for biomedical purposes. Theselivestock gene-editing processes include TALEN- andCRISPR/Cas-stimulated homology-directed repair (HDR) using plasmid, rAAVand oligonucleotide templates. Nucleases such as CRISPR/Cas, TALENs, andzinc finger nucleases are used to target specific nucleic acidsequences. Transcription activator-like (TAL) effector sequences can beassembled to specifically bind DNA targets by assembling sequences ofrepeat variable diresidues (RVDs). Fusion proteins of TAL effectors witha nuclease can make targeted double-stranded breaks in cellular DNA thatcan be used to make specific genetic modifications to cells.

Traditional breeding programs based on animal mating or artificialreproductive techniques involve mixing many genes in the hope ofultimately producing a good combination of genes that create or combinedesirable traits. Transgenic techniques can accelerate traditionalbreeding processes. In some instances, however, transgenic processeswhile perhaps an overall improvement, are nonetheless slow, costly,and/or labor-intensive. Low efficiencies and unpredictability in resultshave slowed some efforts in the field. Further, in traditional breedingprograms, processes that make a change only at a single intended genomicsite are not available.

Gene editing tools such as targeting endonucleases are useful for makinggenetically modified animals. Using these tools to change a nativeallele at only one base is difficult or impossible using conventionalprocesses. New techniques are described herein for making these edits ata single base, or a plurality of single-base edits. These processes areuseful for introgression of an allele that differs only by a singlenucleotide polymorphism (SNP) or a plurality of SNPs. The ability tointrogress SNPs from one breed or species into another is believed tocreate important new opportunities. The term SNP refers to a differenceof one base at the same relative site when two alleles are aligned andcompared; herein, the term is also used in some contexts to mean asingle base change.

Disclosed herein are processes to make transgenic animals that havechanges only at an intended site. Additionally, the processes can makespecifically intended changes at the intended site. In some instances,it is not necessary to remove other changes resulting from problems likethe use of linked-reporter genes, or linked positive and negativeselection genes, or random transgene integration, as the inclusion ofsuch features are bypassed. Moreover, the processes can be used in thefounder generation to make genetically modified animals that have onlythe intended change at the intended site. Other processes are alsodisclosed that involve unlinked marker genes and the like. Someembodiments use TALENs.

Compositions and methods of making higher animals, such as swine orcows, with genetic modifications are set forth herein. Some of thesemethods involve cloning from primary artiodactyl or other livestockcells. Further, methods for identifying cells or embryos that have beenmodified with TALENs are presented, as well as processes for enrichingthe percentage of TALEN-treated cells or embryos. Unexpectedly, it wasobserved that a genetic modification of one chromosome by a TALEN oftencaused the complementary locus of the other chromosome to also bemodified by cellular machinery.

Further, it was also discovered that TALENs could be used to make grosschromosomal deletions (GCDs) at a plurality of sites. FIG. 2 illustratesthis approach, which involves a first TALEN pair directed to a firstlocus and a second TALEN pair directed to a second locus. It was alsosurprisingly discovered that inversions of large chromosomal sequencescould be created using pairs of TALENs. One use of the inversions is thecreation of artiodactyls or other founder animals with fixed genetictraits, or the creation of deletion strains.

Targeted endonuclease technologies, such as zinc finger nucleases(ZFNs), TAL effector nucleases (TALENs) and clustered regularlyinterspaced short palindromic repeats/CRISPR associated endonucleasecas9 (CRISPR/Cas9) can be utilized to disrupt gene-function byintroducing insertions and/or deletions (indels) into genomes ofspecies, such as by non-homologous end-joining (NHEJ). However, indelsintroduced by NHEJ are variable in size and sequence which makesscreening for functionally disrupted clones arduous and does not enableprecise alterations. TALEN or CRISPR/Cas9 mediated homology-directedrepair (HDR) supports the introduction of defined nucleotide changes inlower eukaryotic models including yeast, zebrafish and, very recently,mice. These are models that allow for long-passage cells or primordialgerm cells to be modified to make transgenic animals.

Demonstrated herein is precise, high frequency editing of a variety ofgenes in numerous working examples as exemplified in pig, goat, andcattle genomes. In some embodiments, the gene edits areindistinguishable from alleles that exist within a species or clade andrepresent the first demonstration of marker-free, non-meiotic alleleintrogression. High-efficiency and precise gene editing was achieved incertain commercially important loci in the genomes of livestock that areuseful for agriculture, for research tools, or for biomedical purposes.

These processes have expanded the livestock gene-editing toolbox toinclude TALEN- and CRISPR/Cas9-stimulated homology-directed repair (HDR)using plasmid, rAAV, and oligonucleotide templates. Examples show thatthe bovine POLLED allele was introgressed into horned Holsteinfibroblasts. This example demonstrates that various breeds of dairycattle can be created that do not have horns. And this change can bemade without disturbing other genes, or other parts of the genome, ofthe animals or cells. Single nucleotide alterations or small indels wereintroduced into other genes in pig, goat and cattle fibroblasts usingTALEN mRNA and oligonucleotide transfection with efficiencies of 10-50%in populations. Several of the chosen edits mimicked naturally occurringperformance enhancing or disease resistance alleles including, for thefirst time, alteration of single base pairs (bp). Up to 70% offibroblasts colonies propagated without selection harbored the intendededits, of which over one half were homozygous. These efficiencies aresufficiently high that these changes can be made without reportersand/or without selection markers. These methods demonstrate meiosis-freeintra- and inter-specific introgression of select alleles in livestockcells, large mammals, and livestock for research, agricultural andbiomedical applications.

Sequences that are similar to, but differ somewhat from, the particularsequences described herein may be used, such as for generation ofTALENs, guide RNAs, or homology-dependent repair templates. Forinstance, in some cases, sequences may be used that have at least 80%homology to the sequences described with particularly herein. The term“homology,” as used herein, generally refers to calculations of“homology” or “percent homology” between two or more nucleotide or aminoacid sequences that can be determined by aligning the sequences foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of a first sequence). The nucleotides at correspondingpositions may then be compared, and the percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100). For example, if a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. In some embodiments, thelength of a sequence aligned for comparison purposes is at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%, of the length of the reference sequence. In some cases,a sequence homology may be from about 70% to 100%. In some cases, asequence homology may be from about 80% to 100%. In some cases, asequence homology may be from about 90% to 100%. In some cases, asequence homology may be from about 95% to 100%. In some cases, asequence homology may be from about 80% to 99%. In some cases, asequence homology may be from about 90% to 99%. In some cases, asequence homology may be from about 95% to 99%. A BLAST® search maydetermine homology between two sequences. The two sequences can begenes, nucleotides sequences, protein sequences, peptide sequences,amino acid sequences, or fragments thereof.

Genetically Modified Animals

Animals may be modified using TALENs, zinc finger nucleases, or othergenetic engineering tools, including various vectors that are known. Agenetic modification made by such tools may comprise inactivation of agene. The term inactivation of a gene refers to preventing the formationof a functional gene product. A gene product is functional only if itfulfills its normal (wild-type) functions. Materials and methods ofgenetically modifying animals are further detailed in U.S. Ser. No.13/404,662 filed Feb. 24, 2012, Ser. No. 13/467,588 filed May 9, 2012,and Ser. No. 12/622,886 filed Nov. 10, 2009 which are herebyincorporated herein by reference for all purposes; in case of conflict,the instant specification is controlling. The term trans-acting refersto processes acting on a target gene from a different molecule (i.e.,intermolecular). A trans-acting element is usually a DNA sequence thatcontains a gene. This gene codes for a protein (or microRNA or otherdiffusible molecule) that is used in the regulation of the target gene.The trans-acting gene may be on the same chromosome as the target gene,but the activity is via the intermediary protein or RNA that it encodes.Inactivation of a gene using a dominant negative generally involves atrans-acting element. The term cis-regulatory or cis-acting means anaction without coding for protein or RNA; in the context of geneinactivation, this generally means inactivation of the coding portion ofa gene, or a promoter and/or operator that is necessary for expressionof the functional gene.

Various techniques known in the art can be used to introduce nucleicacid constructs into non-human animals to produce founder lines, inwhich the nucleic acid construct is integrated into the genome. Suchtechniques include, without limitation, pronuclear microinjection (U.S.Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines(Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652),gene targeting into embryonic stem cells (Thompson et al. (1989) Cell56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3,1803-1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc.Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. (2006) Reprod.Fert. Develop. 18, 19-23), and in vitro transformation of somatic cells,such as cumulus or mammary cells, or adult, fetal, or embryonic stemcells, followed by nuclear transplantation (Wilmut et al. (1997) Nature385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374).Pronuclear microinjection, sperm mediated gene transfer, and somaticcell nuclear transfer are particularly useful techniques, as well ascytoplasmic injection, primordial germ cell transplantation (Brinster),and blastocyst chimera production whereby a germ cell is propagated inan embryo.

Typically, in embryo/zygote pronuclear microinjection, a nucleic acidconstruct or mRNA is introduced into a fertilized egg; 1 or 2 cellfertilized eggs are used as the pronuclei containing the geneticmaterial from the sperm head and the egg are visible within theprotoplasm. Pronuclear staged fertilized eggs can be obtained in vitroor in vivo (i.e., surgically recovered from the oviduct of donoranimals). In vitro fertilized eggs can be produced as follows. Forexample, swine ovaries can be collected at an abattoir, and maintainedat 22-28° C. during transport. Ovaries can be washed and isolated forfollicular aspiration, and follicles ranging from 4-8 mm can beaspirated into 50 mL conical centrifuge tubes using 18 gauge needles andunder vacuum. Follicular fluid and aspirated oocytes can be rinsedthrough pre-filters with commercial TL-HEPES (Minitube, Verona, Wis.).Oocytes surrounded by a compact cumulus mass can be selected and placedinto TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, Wis.)supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor,10% porcine follicular fluid, 50 μM 2-mercaptoethanol, 0.5 mg/ml cAMP,10 IU/mL each of pregnant mare serum gonadotropin (PMSG) and humanchorionic gonadotropin (hCG) for approximately 22 hours in humidifiedair at 38.7° C. and 5% CO2. Subsequently, the oocytes can be moved tofresh TCM-199 maturation medium, which will not contain cAMP, PMSG orhCG and incubated for an additional 22 hours. Matured oocytes can bestripped of their cumulus cells by vortexing in 0.1% hyaluronidase for 1minute.

For swine, mature oocytes can be fertilized in 500 μl Minitube PORCPROIVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in Minitube 5-wellfertilization dishes. In preparation for in vitro fertilization (IVF),freshly-collected or frozen boar semen can be washed and resuspended inPORCPRO IVF Medium to 4×105 sperm. Sperm concentrations can be analyzedby computer assisted semen analysis (SPERMVISION, Minitube, Verona,Wis.). Final in vitro insemination can be performed in a 10 μl volume ata final concentration of approximately 40 motile sperm/oocyte, dependingon boar. Incubate all fertilizing oocytes at 38.7° C. in 5.0% CO₂atmosphere for 6 hours. Six hours post-insemination, presumptive zygotescan be washed twice in NCSU-23 and moved to 0.5 mL of the same medium.This system can produce 20-30% blastocysts routinely across most boarswith a 10-30% polyspermic insemination rate. Linearized nucleic acidconstructs can be injected into one of the pronuclei, or e.g.,transposons or cytoplasmic injection may be used. Then the injected eggscan be transferred to a recipient female (e.g., into the oviducts of arecipient female) and allowed to develop in the recipient female toproduce the transgenic animals. In particular, in vitro fertilizedembryos can be centrifuged at 15,000×g for 5 minutes to sediment lipidsallowing visualization of the pronucleus. The embryos can be injectedwith using an Eppendorf FEMTOJET injector and can be cultured untilblastocyst formation. Rates of embryo cleavage and blastocyst formationand quality can be recorded.

Embryos can be surgically transferred into uteri of asynchronousrecipients. Typically, 100-200 (e.g., 150-200) embryos can be depositedinto the ampulla-isthmus junction of the oviduct using a 5.5-inchTOMCAT® catheter. After surgery, real-time ultrasound examination ofpregnancy can be performed.

In somatic cell nuclear transfer, a transgenic artiodactyl cell (e.g., atransgenic pig cell or bovine cell) such as an embryonic blastomere,fetal fibroblast, adult ear fibroblast, or granulosa cell that includesa nucleic acid construct described above, can be introduced into anenucleated oocyte to establish a combined cell. Oocytes can beenucleated by partial zona dissection near the polar body and thenpressing out cytoplasm at the dissection area. Typically, an injectionpipette with a sharp beveled tip is used to inject the transgenic cellinto an enucleated oocyte arrested at meiosis 2. In some conventions,oocytes arrested at meiosis-2 are termed “eggs.” After producing aporcine or bovine embryo (e.g., by fusing and activating the oocyte),the embryo is transferred to the oviducts of a recipient female, about20 to 24 hours after activation. See, for example, Cibelli et al. (1998)Science 280, 1256-1258 and U.S. Pat. No. 6,548,741. For pigs, recipientfemales can be checked for pregnancy approximately 20-21 days aftertransfer of the embryos.

Standard breeding techniques can be used to create animals that arehomozygous for the target nucleic acid from the initial heterozygousfounder animals. Homozygosity may not be required in some instances,however. Transgenic pigs described herein can be bred with other pigs ofinterest.

In some embodiments, a nucleic acid of interest and a selectable markercan be provided on separate transposons and provided to either embryosor cells in unequal amount, where the amount of transposon containingthe selectable marker far exceeds (5-10 fold excess) the transposoncontaining the nucleic acid of interest. Transgenic cells or animalsexpressing the nucleic acid of interest can be isolated based onpresence and expression of the selectable marker. Because thetransposons will integrate into the genome in a precise and unlinked way(independent transposition events), the nucleic acid of interest and theselectable marker are not genetically linked and can easily be separatedby genetic segregation through standard breeding. Thus, transgenicanimals can be produced that are not constrained to retain selectablemarkers in subsequent generations, an issue of some concern from apublic safety perspective.

Once transgenic animal have been generated, expression of a targetnucleic acid can be assessed using standard techniques. Initialscreening can be accomplished by Southern blot analysis to determinewhether or not integration of the construct has taken place. For adescription of Southern analysis, see sections 9.37-9.52 of Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, second edition, ColdSpring Harbor Press, Plainview; NY. Polymerase chain reaction (PCR)techniques also can be used in the initial screening. PCR refers to aprocedure or technique in which target nucleic acids are amplified.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of the template tobe amplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. PCRis described in, for example PCR Primer: A Laboratory Manual, ed.Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.Nucleic acids also can be amplified by ligase chain reaction, stranddisplacement amplification, self-sustained sequence replication, ornucleic acid sequence-based amplified. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874; and Weiss (1991) Science 254:1292. At the blastocyststage, embryos can be individually processed for analysis by, e.g., PCR,Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al.Proc Natl Acad Sci USA (2002) 99:4495).

Expression of a nucleic acid sequence encoding a polypeptide in thetissues of transgenic pigs can be assessed using techniques thatinclude, for example, Northern blot analysis of tissue samples obtainedfrom the animal, in situ hybridization analysis, Western analysis,immunoassays such as enzyme-linked immunosorbent assays, andreverse-transcriptase PCR (RT-PCR).

Founder Animals, Animals Lines, Traits and Reproduction

Founder animals may be produced by cloning and other methods describedherein. The founders can be homozygous for a genetic modification, as inthe case where a zygote or a primary cell undergoes a homozygousmodification. Similarly, founders can also be made that areheterozygous. The founders may be genomically modified, meaning that allof the cells in their genome have undergone modification. Founders canbe mosaic for a modification, as may happen when vectors are introducedinto one of a plurality of cells in an embryo, typically at a blastocyststage. Progeny of mosaic animals may be tested to identify progeny thatare genomically modified. An animal line is established when a pool ofanimals has been created that can be reproduced sexually or by assistedreproductive techniques, with heterogeneous or homozygous progenyconsistently expressing the modification.

In livestock, many alleles are known to be linked to various traits suchas production traits, type traits, workability traits, and otherfunctional traits. Artisans are accustomed to monitoring and quantifyingthese traits, e.g., Visscher et al., Livestock Production Science, 40(1994) 123-137, U.S. Pat. No. 7,709,206, US 2001/0016315, US2011/0023140, and US 2005/0153317. An animal line may include a traitchosen from a trait in the group consisting of a production trait, atype trait, a workability trait, a fertility trait, a mothering trait,and a disease resistance trait. Further traits include expression of arecombinant gene product.

Animals with a desired trait or traits may be modified to prevent theirsexual maturation. Since the animals are sterile until matured, it ispossible to regulate sexual maturity as a means of controllingdissemination of the animals. Animals that have been bred or modified tohave one or more traits can thus be provided to recipients with areduced risk that the recipients will breed the animals and appropriatethe value of the traits to themselves. Embodiments of the inventioninclude genetically modifying a genome of an animal with themodification comprising an inactivated sexual maturation gene, whereinthe sexual maturation gene in a wild type animal expresses a factorselective for sexual maturation. Embodiments include treating the animalby administering a compound to remedy a deficiency caused by the loss ofexpression of the gene to induce sexual maturation in the animal.

Breeding of animals that require administration of a compound to inducesexual maturity may advantageously be accomplished at a treatmentfacility. The treatment facility can implement standardized protocols onwell-controlled stock to efficiently produce consistent animals. Theanimal progeny may be distributed to a plurality of locations to beraised. Farms and farmers (a term including a ranch and ranchers) maythus order a desired number of progeny with a specified range of agesand/or weights and/or traits and have them delivered at a desired timeand/or location. The recipients, e.g., farmers, may then raise theanimals and deliver them to market as they desire.

Embodiments include delivering (e.g., to one or more locations, to aplurality of farms) a genetically modified livestock animal having aninactivated neuroendocrine gene selective for sexual maturation.Embodiments include delivery of animals having an age of between about 1day and about 180 days. The animal may have one or more traits (forexample one that expresses a desired trait or a high-value trait or anovel trait or a recombinant trait). Embodiments further includeproviding said animal and/or breeding said animal.

Polypeptides

There are a variety of conservative changes that can generally be madeto an amino acid sequence without altering activity. These changes aretermed conservative substitutions or mutations; that is, an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic can be substituted for another amino acid. Substitutesfor an amino acid sequence may be selected from other members of theclass to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and tyrosine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Such alterationsare not expected to substantially affect apparent molecular weight asdetermined by polyacrylamide gel electrophoresis or isoelectric point.Exemplary conservative substitutions include, but are not limited to,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that afree——OH is maintained; and Gln for Asn to maintain a free NH₂.Moreover, point mutations, deletions, and insertions of the polypeptidesequences or corresponding nucleic acid sequences may in some cases bemade without a loss of function of the polypeptide or nucleic acidfragment. Substitutions may include, e.g., 1, 2, 3, or more residues.The amino acid residues described herein employ either the single letteramino acid designator or the three-letter abbreviation. Abbreviationsused herein are in keeping with the standard polypeptide nomenclature,J. Biol. Chem., (1969), 243, 3552-3559. All amino acid residue sequencesare represented herein by formulae with left and right orientation inthe conventional direction of amino-terminus to carboxy-terminus.

In some cases a determination of the percent identity of a peptide to asequence set forth herein may be required. In such cases, the percentidentity is measured in terms of the number of residues of the peptide,or a portion of the peptide. A polypeptide of, e.g., 90% identity, mayalso be a portion of a larger peptide. Embodiments include suchpolypeptides that have the indicated identity and/or conservativesubstitution of sequence set forth herein.

The term purified as used herein with reference to a polypeptide refersto a polypeptide that either has no naturally occurring counterpart(e.g., a peptidomimetic), or has been chemically synthesized and is thussubstantially uncontaminated by other polypeptides, or has beenseparated or purified from other most cellular components by which it isnaturally accompanied (e.g., other cellular proteins, polynucleotides,or cellular components). An example of a purified polypeptide is onethat is at least 70%, by dry weight, free from the proteins andnaturally occurring organic molecules with which it naturallyassociates. A preparation of a purified polypeptide therefore can be,for example, at least 80%, at least 90%, or at least 99%, by dry weight,the polypeptide. Polypeptides also can be engineered to contain a tagsequence (e.g., a polyhistidine tag, a myc tag, or a FLAG® tag) thatfacilitates the polypeptide to be purified or marked (e.g., capturedonto an affinity matrix, visualized under a microscope). Thus a purifiedcomposition that comprises a polypeptide refers to a purifiedpolypeptide unless otherwise indicated.

Polypeptides may include a chemical modification; a term that, in thiscontext, refers to a change in the naturally-occurring chemicalstructure of amino acids. Such modifications may be made to a side chainor a terminus, e.g., changing the amino-terminus or carboxyl terminus.In some embodiments, the modifications are useful for creating chemicalgroups that may conveniently be used to link the polypeptides to othermaterials, or to attach a therapeutic agent.

Interfering RNAs

A variety of interfering RNA (RNAi) are known. Double-stranded RNA(dsRNA) induces sequence-specific degradation of homologous genetranscripts. RNA-induced silencing complex (RISC) metabolizes dsRNA tosmall 21-23-nucleotide small interfering RNAs (siRNAs). RISC contains adouble stranded RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut2 or Ago2). RISC utilizes antisense strand as a guide to find acleavable target. Both siRNAs and microRNAs (miRNAs) are known. A methodof inactivating a gene in a genetically modified animal comprisesinducing RNA interference against a target gene and/or nucleic acid suchthat expression of the target gene and/or nucleic acid is reduced.

For example the exogenous nucleic acid sequence can induce RNAinterference against a nucleic acid encoding a polypeptide. For example,double-stranded small interfering RNA (siRNA) or small hairpin RNA(shRNA) homologous to a target DNA can be used to reduce expression ofthat DNA. Constructs for siRNA can be produced as described, forexample, in Fire et al. (1998) Nature 391:806; Romano and Masino (1992)Mol. Microbiol. 6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni andMasino (1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl.Acad. Sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell 95:1017.Constructs for shRNA can be produced as described by McIntyre andFanning (2006) BMC Biotechnology 6:1. In general, shRNAs are transcribedas a single-stranded RNA molecule containing complementary regions,which can anneal and form short hairpins.

The probability of finding a single, individual functional siRNA ormiRNA directed to a specific gene is high. The predictability of aspecific sequence of siRNA, for instance, is about 50% but a number ofinterfering RNAs may be made with good confidence that at least one ofthem will be effective.

Embodiments include an in vitro cell, an in vivo cell, and a geneticallymodified animal such as a livestock animal that express an RNAi directedagainst a neuroendocrine gene selective for sexual maturation. Anembodiment is an RNAi directed against a gene in the group consisting ofGpr54, Kiss1, and GnRH1. The RNAi may be, for instance, selected fromthe group consisting of siRNA, shRNA, dsRNA, RISC and miRNA.

Vectors and Nucleic Acids

A variety of nucleic acids may be introduced into the artiodactyl orother cells, for knockout purposes, or to obtain expression of a genefor other purposes. Nucleic acid constructs that can be used to producetransgenic animals include a target nucleic acid sequence. As usedherein, the term nucleic acid includes DNA, RNA, and nucleic acidanalogs, and nucleic acids that are double-stranded or single-stranded(i.e., a sense or an antisense single strand). Nucleic acid analogs canbe modified at the base moiety, sugar moiety, or phosphate backbone toimprove, for example, stability, hybridization, or solubility of thenucleic acid. Modifications at the base moiety include deoxyuridine fordeoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. See,Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3):187;and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5. In addition, thedeoxyphosphate backbone can be replaced with, for example, aphosphorothioate or phosphorodithioate backbone, a phosphoroamidite, oran alkyl phosphotriester backbone.

The target nucleic acid sequence can be operably linked to a regulatoryregion such as a promoter. Regulatory regions can be porcine regulatoryregions or can be from other species. As used herein, operably linkedrefers to positioning of a regulatory region relative to a nucleic acidsequence in such a way as to permit or facilitate transcription of thetarget nucleic acid.

Any type of promoter can be operably linked to a target nucleic acidsequence. Examples of promoters include, without limitation,tissue-specific promoters, constitutive promoters, and promotersresponsive or unresponsive to a particular stimulus. Suitable tissuespecific promoters can result in preferential expression of a nucleicacid transcript in beta cells and include, for example, the humaninsulin promoter. Other tissue specific promoters can result inpreferential expression in, for example, hepatocytes or heart tissue andcan include the albumin or alpha-myosin heavy chain promoters,respectively. In other embodiments, a promoter that facilitates theexpression of a nucleic acid molecule without significant tissue- ortemporal-specificity can be used (i.e., a constitutive promoter). Forexample, a beta-actin promoter such as the chicken beta-actin genepromoter, ubiquitin promoter, miniCAGs promoter,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or3-phosphoglycerate kinase (PGK) promoter can be used, as well as viralpromoters such as the herpes simplex virus thymidine kinase (HSV-TK)promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter. Insome embodiments, a fusion of the chicken beta actin gene promoter andthe CMV enhancer is used as a promoter. See, for example, Xu et al.(2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther.7:821.

An example of an inducible promoter is the tetracycline (tet)-onpromoter system, which can be used to regulate transcription of thenucleic acid. In this system, a mutated Tet repressor (TetR) is fused tothe activation domain of herpes simplex virus VP16 trans-activatorprotein to create a tetracycline-controlled transcriptional activator(tTA), which is regulated by tet or doxycycline (dox). In the absence ofantibiotic, transcription is minimal, while in the presence of tet ordox, transcription is induced. Alternative inducible systems include theecdysone or rapamycin systems. Ecdysone is an insect molting hormonewhose production is controlled by a heterodimer of the ecdysone receptorand the product of the ultraspiracle gene (USP). Expression is inducedby treatment with ecdysone or an analog of ecdysone such as muristeroneA. The agent that is administered to the animal to trigger the induciblesystem is referred to as an induction agent.

Additional regulatory regions that may be useful in nucleic acidconstructs, include, but are not limited to, polyadenylation sequences,translation control sequences (e.g., an internal ribosome entry segment,IRES), enhancers, inducible elements, or introns. Such regulatoryregions may not be necessary, although they may increase expression byaffecting transcription, stability of the mRNA, translationalefficiency, or the like. Such regulatory regions can be included in anucleic acid construct as desired to obtain optimal expression of thenucleic acids in the cell(s). Sufficient expression, however, cansometimes be obtained without such additional elements.

A nucleic acid construct may be used that encodes signal peptides orselectable markers. Signal peptides can be used such that an encodedpolypeptide is directed to a particular cellular location (e.g., thecell surface). Non-limiting examples of selectable markers includepuromycin, ganciclovir, adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR),hygromycin-B-phosphtransferase, thymidine kinase (TK), andxanthin-guanine phosphoribosyltransferase (XGPRT). Such markers areuseful for selecting stable transformants in culture. Other selectablemarkers include fluorescent polypeptides, such as green fluorescentprotein or yellow fluorescent protein.

In some embodiments, a sequence encoding a selectable marker can beflanked by recognition sequences for a recombinase such as, e.g., Cre orFlp. For example, the selectable marker can be flanked by loxPrecognition sites (34-bp recognition sites recognized by the Crerecombinase) or FRT recognition sites such that the selectable markercan be excised from the construct. See, Orban, et al., Proc. Natl. Acad.Sci. (1992) 89:6861, for a review of Cre/lox technology, and Brand andDymecki, Dev. Cell (2004) 6:7. A transposon containing a Cre- orFlp-activatable transgene interrupted by a selectable marker gene alsocan be used to obtain transgenic animals with conditional expression ofa transgene. For example, a promoter driving expression of themarker/transgene can be either ubiquitous or tissue-specific, whichwould result in the ubiquitous or tissue-specific expression of themarker in F0 animals (e.g., pigs). Tissue specific activation of thetransgene can be accomplished, for example, by crossing a pig thatubiquitously expresses a marker-interrupted transgene to a pigexpressing Cre or Flp in a tissue-specific manner, or by crossing a pigthat expresses a marker-interrupted transgene in a tissue-specificmanner to a pig that ubiquitously expresses Cre or Flp recombinase.Controlled expression of the transgene or controlled excision of themarker allows expression of the transgene.

In some embodiments, the target nucleic acid encodes a polypeptide. Anucleic acid sequence encoding a polypeptide can include a tag sequencethat encodes a “tag” designed to facilitate subsequent manipulation ofthe encoded polypeptide (e.g., to facilitate localization or detection).Tag sequences can be inserted in the nucleic acid sequence encoding thepolypeptide such that the encoded tag is located at either the carboxylor amino terminus of the polypeptide. Non-limiting examples of encodedtags include glutathione S transferase (GST) and FLAG™ tag (Kodak, NewHaven, Conn.).

In other embodiments, the target nucleic acid sequence induces RNAinterference against a target nucleic acid such that expression of thetarget nucleic acid is reduced. For example the target nucleic acidsequence can induce RNA interference against a nucleic acid encoding acystic fibrosis transmembrane conductance regulatory (CFTR) polypeptide.For example, double-stranded small interfering RNA (siRNA) or shorthairpin RNA (shRNA) homologous to a CFTR DNA can be used to reduceexpression of that DNA. Constructs for siRNA can be produced asdescribed, for example, in Fire et al. (1998) Nature 391:806; Romano andMasino (1992) Mol. Microbiol. 6:3343; Cogoni et al. (1996) EMBO J.15:3153; Cogoni and Masino (1999) Nature 399:166; Misquitta and Paterson(1999) Proc. Natl. Acad. Sci. USA 96:1451; and Kennerdell and Carthew(1998) Cell 95:1017. Constructs for shRNA can be produced as describedby McIntyre and Fanning (2006) BMC Biotechnology 6:1. In general, shRNAsare transcribed as a single-stranded RNA molecule containingcomplementary regions, which can anneal and form short hairpins.

Nucleic acid constructs can be methylated using an SssI CpG methylase(New England Biolabs, Ipswich, Mass.). In general, the nucleic acidconstruct can be incubated with S-adenosylmethionine and SssICpG-methylase in buffer at 37° C. Hypermethylation can be confirmed byincubating the construct with one unit of HinP1I endonuclease for 1 hourat 37° C. and assaying by agarose gel electrophoresis.

Nucleic acid constructs can be introduced into embryonic, fetal, oradult artiodactyl cells of any type, including, for example, germ cellssuch as an oocyte or an egg, a progenitor cell, an adult or embryonicstem cell, a primordial germ cell, a kidney cell such as a PK-15 cell,an islet cell, a beta cell, a liver cell, or a fibroblast such as adermal fibroblast, using a variety of techniques. Non-limiting examplesof techniques include the use of transposon systems, recombinant virusesthat can infect cells, or liposomes or other non-viral methods such aselectroporation, microinjection, or calcium phosphate precipitation,that are capable of delivering nucleic acids to cells.

In transposon systems, the transcriptional unit of a nucleic acidconstruct, i.e., the regulatory region operably linked to a targetnucleic acid sequence, is flanked by an inverted repeat of a transposon.Several transposon systems, including, for example, Sleeping Beauty(see, U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542);Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2(Kawakami (2007) Genome Biology 8(Suppl.1):S7; Minos (Pavlopoulos et al.(2007) Genome Biology 8(Suppl.1):S2); Hsmar1 (Miskey et al. (2007)) MolCell Biol. 27:4589); and Passport have been developed to introducenucleic acids into cells, including mice, human, and pig cells. TheSleeping Beauty and Passport transposon is particularly useful. Atransposase can be delivered as a protein, encoded on the same nucleicacid construct as the target nucleic acid, can be introduced on aseparate nucleic acid construct, or provided as an mRNA (e.g., an invitro-transcribed and capped mRNA).

Insulator elements also can be included in a nucleic acid construct tomaintain expression of the target nucleic acid and to inhibit theunwanted transcription of host genes. See, for example, U.S. PublicationNo. 2004/0203158. Typically, an insulator element flanks each side ofthe transcriptional unit and is internal to the inverted repeat of thetransposon. Non-limiting examples of insulator elements include thematrix attachment region-(MAR) type insulator elements and border-typeinsulator elements. See, for example, U.S. Pat. Nos. 6,395,549,5,731,178, 6,100,448 and 5,610,053, and U.S. Publication No.2004/0203158.

Nucleic acids can be incorporated into vectors. A vector is a broad termthat includes any specific DNA segment that is designed to move from acarrier into a target DNA. A vector may be referred to as an expressionvector, or a vector system, which is a set of components needed to bringabout DNA insertion into a genome or other targeted DNA sequence such asan episome, plasmid, or even virus/phage DNA segment. Vector systemssuch as viral vectors (e.g., retroviruses, adeno-associated virus andintegrating phage viruses), and non-viral vectors (e.g., transposons)used for gene delivery in animals have two basic components: 1) a vectorcomprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2)a transposase, recombinase, or other integrase enzyme that recognizesboth the vector and a DNA target sequence and inserts the vector intothe target DNA sequence. Vectors most often contain one or moreexpression cassettes that comprise one or more expression controlsequences, wherein an expression control sequence is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence or mRNA, respectively.

Many different types of vectors are known. For example, plasmids andviral vectors, e.g., retroviral vectors, are known. Mammalian expressionplasmids typically have an origin of replication, a suitable promoterand optional enhancer, and also any necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences.Examples of vectors include: plasmids (which may also be a carrier ofanother type of vector), adenovirus, adeno-associated virus (AAV),lentivirus (e.g., HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV orMoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, FrogPrince, piggyBac).

As used herein, the term nucleic acid refers to both RNA and DNA,including, for example, cDNA, genomic DNA, synthetic (e.g., chemicallysynthesized) DNA, as well as naturally occurring and chemically modifiednucleic acids, e.g., synthetic bases or alternative backbones. A nucleicacid molecule can be double-stranded or single-stranded (i.e., a senseor an antisense single strand). The term transgenic is used broadlyherein and refers to a genetically modified organism or geneticallyengineered organism whose genetic material has been altered usinggenetic engineering techniques. A knockout artiodactyl is thustransgenic regardless of whether or not exogenous genes or nucleic acidsare expressed in the animal or its progeny.

The nucleic acid sequences set forth herein are intended to representboth DNA and RNA sequences, according to the conventional practice ofallowing the abbreviation “T” stand for “T” or for “U”, as the case maybe, for DNA or RNA. Polynucleotides are nucleic acid molecules of atleast three nucleotide subunits. Polynucleotide analogues or polynucleicacids are chemically modified polynucleotides or polynucleic acids. Insome embodiments, polynucleotide analogues can be generated by replacingportions of the sugar-phosphate backbone of a polynucleotide withalternative functional groups. Morpholino-modified polynucleotides,referred to herein as “morpholinos,” are polynucleotide analogues inwhich the bases are linked by a morpholino-phosphorodiamidate backbone(see, e.g., U.S. Pat. Nos. 5,142,047 and 5,185,444). In addition tomorpholinos, other examples of polynucleotide analogues includeanalogues in which the bases are linked by a polyvinyl backbone, peptidenucleic acids (PNAs) in which the bases are linked by amide bonds formedby pseudopeptide 2-aminoethyl-glycine groups, analogues in which thenucleoside subunits are linked by methylphosphonate groups, analogues inwhich the phosphate residues linking nucleoside subunits are replaced byphosphoroamidate groups, and phosphorothioated DNAs, analoguescontaining sugar moieties that have 2′ O-methyl group). Polynucleotidesof the invention can be produced through the well-known and routinelyused technique of solid phase synthesis. Alternatively, other suitablemethods for such synthesis can be used (e.g., common molecular cloningand chemical nucleic acid synthesis techniques). Similar techniques alsocan be used to prepare polynucleotide analogues such as morpholinos orphosphorothioate derivatives. In addition, polynucleotides andpolynucleotide analogues can be obtained commercially. Foroligonucleotides, examples of pharmaceutically acceptable compositionsare salts that include, e.g., (a) salts formed with cations such assodium, potassium, ammonium, etc.; (b) acid addition salts formed withinorganic acids, for example, hydrochloric acid, hydrobromic acid (c)salts formed with organic acids e.g., for example, acetic acid, oxalicacid, tartaric acid; and (d) salts formed from elemental anions e.g.,chlorine, bromine, and iodine.

A sequence alignment is a way of arranging the sequences of DNA, RNA, orprotein to identify regions of similarity. Aligned sequences ofnucleotide or amino acid residues are typically represented as rowswithin a matrix, with gaps are inserted between the residues so thatidentical or similar characters are aligned in successive columns.

Dominant Negatives

Genes may thus be inactivated not only by removal or RNAi suppressionbut also by creation of a dominant negative phenotype. A dominantnegative version of a gene product lacks one or more functions of thewild-type phenotype and dominantly interferes with the function of anormal gene product expressed in the same cell, with a result that thedominant negative phenotype effectively decreases or inactivates thephysiological outcome normally expected to be elicited by a gene'snormal function. For example, the function of most proteins requirestheir interaction with other proteins. Such interactions are oftenrequired for proper protein localization, ligand binding, proteinactivation, or the downstream transduction of upstream signals. Themutation of one or more of the components of a multi-protein complex caninterfere with one these processes. Thus, the expression of a mutantform of a protein can interfere with a proteins function, even in thepresence of a normal gene product, acting as a poison “pill” or a“monkey wrench” into the gearbox. GPCRs are seven-transmembrane (7TM)domain receptors which are trafficked through the biosynthetic pathwayto the cell surface in a tightly regulated mechanism with multiple stepsand a stringent quality control system to ensure correct GPCR foldingand targeting. Association of GPCRs with accessory proteins orchaperones are a key step for the forward trafficking through theendoplasmic reticulum (ER) and Golgi. The life of GPCRs begins in the ERwhere they are synthesized, folded and assembled. During their migrationto the cell surface, GPCRs undergo post-translational modifications toattain mature status. Because the ER forms part of the cellular qualitycontrol machinery where functionally inactive mutant GPCRs can beprevented from expression at the cell surface.

Conditions such as X-linked nephrogenic-diabetes insipidus, familialhypocalciuric hypercalcemia, familial glucocorticoid deficiency orhypogonadodotropic hypogonadism are associated with mutations in GPCRswhich result in intracellular retention in the ER or Golgi compartments.In numerous cases the defect in cell surface membrane expression is dueto intracellular association of receptors, with a dominant-negative (DN)effect of the misfolded receptor on its wild-type counterpart; this DNeffect may limit, or even abrogate, plasma membrane expression of thenormal receptor and thus provoke a loss-of-function disease(Ulloa-Aguirre et al., 2004a).

Loss-of-function mutations in the GnRHR can lead to partial or completehypogonadotropic hypogonadism (HH), a failure of pituitary gonadotropesto respond to GnRH, which results in decreased or apulsatilegonadotropin release and reproductive failure. A large number ofmutations leading to receptor misfolding and resultant misrouting of thegonadotropin hormone-releasing hormone receptor (GnRHR) in patients withHH have been described (Janovick et al., 2002; Leaños-Miranda et al.,2002; Ulloa-Aguirre et al., 2004b). Many of these mutations act asDominant negatives for GnRHR function (Pask A J et al, 2005 MolEndocrinol; Brothers S P et al, 2004 Mol Endocrinol; Leaños-Miranda A etal, 2003 J Clin Endocrinol Metab). Thus, purposeful expression of a DNGnRHR gene is expected to cause sterility in transgenic animals.

As discussed, GPR54 is a gatekeeper of the reproductive cascade thatinitiates puberty. Animal studies demonstrate that engagement of GPR54by endogenous peptide ligands, termed kisspeptins, potently stimulatesgonadotropin-releasing hormone release from hypothalamic neurons toactivate the hypothalamic-pituitary-gonadal axis. Furthermore, thecharacterization of GPR54 KO mice, which phenocopy the human conditionof idiopathic hypogonadotropic hypogonadism, confirmed the essentialrole of GPR54 for reproductive function. GPCRs are now recognized toexist as multiprotein complexes composed of GPCR-interacting proteins(GIPs) that impart precise spatial and temporal regulation ofexpression, trafficking, ligand binding, and signaling. GPR54 has beendetermined to specifically interact with these GIPs. Because themajority of truncated GPCR splice variants act as dominant-negativemutations (Wise 2012, J Mol Signal), the expression of GPR54 lacking oneor more transmembrane domains is expected to disrupt theprocessing/trafficking of endogenous GPR54, thus interfering with itsfunction. Thus, purposeful expression of a DN GPR54 gene is expected tocause sterility in transgenic animals.

Templated and Non-Templated Repairs

TALENs, zinc finger nucleases, CRISPR nuclease (e.g., CRISPR/Cas9) andrecombinase fusion proteins may be used with or without a template. Atemplate is an exogenous DNA added to the cell for cellular repairmachinery to use as a guide (template) to repair double stranded breaks(DSB) in DNA. This process is generally referred to as homology directedrepair (HDR). Processes without a template involve making DSBs andproviding for cellular machinery to make repairs that are often lessthan perfect, so that an insertion or deletion (an indel) is made. Thecellular pathway referred to as non-homologous end joining (NHEJ)typically mediates non-templated repairs of DSBs. The term NHEJ iscommonly used to refer to all such non-templated repairs regardless ofwhether the NHEJ was involved, or an alternative cellular pathway.

Extended Hypothermia for Template-Directed Repair

Experiments surprisingly showed that an extended period of hypothermicculture could enhance the efficiency of templating processes.Hypothermic cell cultures are known to be useful for up to about threedays to introduce double-stranded DNA breaks. Conventional theories forthis effect revolve around the idea that the active enzymes are beingdiluted or the DNA is stabilized by inhibiting division.

The data herein, however, are not consistent with these other theories.Instead, it is believed that hypothermia minimizes re-repair of alteredchromosomes as guided by the sister chromatid. In other words, even ifthere is successful integration, the cell may use the sister chromatidat the altered site to undo the changed allele. Moreover, these data arethe first to show that hypothermia could be used to impact templatingprocesses. A surprising aspect of the experiments was that the extendedhypothermic culture did not improve the efficiency of copying thetemplate into the cellular DNA. What it improved was the stability ofthe exogenous allele after it had been copied. In fact, this processalmost tripled the level of SNP HDR-edited alleles.

An embodiment is a hypothermic method of template-directed repair tochange a chromosomal DNA of a cell, comprising introducing into a livingcell a targeted nuclease system and a nucleic acid template, wherein thetargeted nuclease system and the template operate to alter thechromosomal DNA to have identity to the template sequence wherein theliving cell is maintained at a hypothermic culturing temperature below aphysiological temperature for a time period. The length of the culturecan be varied as appropriate, e.g., more than 3 days to 31 days or 72 to800 hours; artisans will immediately appreciate that all ranges andvalues within the explicitly stated range are contemplated; e.g., 72 to80 hours, 80 to 600 hours, 3 days to 5 days, 4 days to 15 days, 3.1 daysto two weeks, and so forth. Extended culture times at about 20° C. havebeen successful (data not shown). The hypothermic culture temperatureranges from 20 to 34° C.; artisans will immediately appreciate that allranges and values within the explicitly stated range are contemplated;e.g., 20 to 25° C., 21 to 26° C., 22 to 27° C., 23 to 28° C., 24 to 29°C., 21 to 30° C. Moreover embodiments include maintaining the culture ata specific temperature within the range as well as allowing the culturetemperature to change while remaining within the range. The term “keptwithin a range” in this context includes both these embodiments.Embodiments include culturing to provide a stability of an alleleintroduced into a cell; for example, a modified allele may remain stable(i.e., continue to be present in the population) for more than 5 celldivisions, or at least 3 cell divisions, or a value between 3 and 10cell divisions; artisans will immediately appreciate that all ranges andvalues within the explicitly stated range are contemplated.

SNPs

These experimental results provide a process for placing singlenucleotide polymorphisms (SNPs) into chromosomal DNA. The SNPs can beplaced at a predetermined position. This control over placement iswithout precedent. For instance an SNP can be placed into an endogenousallele without other SNPs or modifications at other locations. Moreover,and crucially, an endogenous allele can be replaced with an exogenousallele that differs by only one SNP. An endogenous allele can be editedto another allele by the creation of an SNP within the allele. And thereplacements are made with minimal alterations to chromosomal DNA at anylocation in genome of the cell. One or more SNPs may be introgressed.

An embodiment is a method of creating a single nucleotide polymorphism(SNP) in a chromosomal DNA of a cell, comprising introducing a targetednuclease system and a HDR template into the cell, with the targetednuclease system comprising a DNA-binding member for specifically bindingan endogenous cognate sequence in the chromosomal DNA, wherein thetargeted nuclease system and the HDR template operate to alter thechromosomal DNA to have identity to the HDR template sequence, whereinthe HDR template sequence comprises a SNP. The HDR template may have aplurality of SNPs or only one. Other changes may be present, e.g.,insertions, deletions, or substitutions. Or the changes may be limitedto a single SNP, or one or a plurality of SNPs introgressed into theendogenous allele. The HDR template sequence may comprise an exogenousallele that replaces an endogenous allele, with the exogenous allelecomprising an SNP in a sequence alignment with the endogenous allele.

Further embodiments include placing an SNP into a cognate site for aDNA-binding member of a targeted nuclease system. The SNP may be chosento reduce binding to the DNA-binding member. One SNP may be thuslyplaced, or a plurality. Further changes, SNPs, or others, may be presentin the allele, or not. The chromosomal DNA may be free of all otherchanges.

Embodiments include a genetically modified animal, the animal belongingto a breed of animals having an endogenous allele, the animal comprisinga genetic change at an SNP to change the chromosomal DNA of the animalfrom the endogenous allele to an exogenous allele found in anotherspecies or another breed of animal. The animal may comprise one or moreof: a plurality of SNPs to change the chromosomal DNA of the animal fromthe endogenous allele to an exogenous allele found in another species oranother breed of animal; further being free or reporters; beinghomozygous for the polymorphism, SNP or SNPs; being a livestock,primate, swine, cow, horse, sheep, goat, avian, chicken, rabbit, fish,dog, mouse, cat, rat, and laboratory animal.

These various embodiments can be performed in a reporter-free system andto make an SNP or an embodiment relating to an SNP. The cells or animalsmay be, e.g., livestock, primate, swine, cow, horse, sheep, goat, avian,chicken, rabbit, fish, dog, mouse, cat, rat, and laboratory animal.

Targeted Nuclease Systems

Genome editing tools such as transcription activator-like effectornucleases (TALENs) and zinc finger nucleases (ZFNs) have impacted thefields of biotechnology, gene therapy and functional genomic studies inmany organisms. More recently, RNA-guided endonucleases (RGENs) aredirected to their target sites by a complementary RNA molecule. TheCas9/CRISPR system is a RGEN. tracrRNA is another such tool. These areexamples of targeted nuclease systems: these system have a DNA-bindingmember that localizes the nuclease to a target site. The site is thencut by the nuclease. TALENs and ZFNs have the nuclease fused to theDNA-binding member. Cas9/CRISPR are cognates that find each other on thetarget DNA. The DNA-binding member has a cognate sequence in thechromosomal DNA. The DNA-binding member is typically designed in lightof the intended cognate sequence so as to obtain a nucleolytic action atnor near an intended site. Certain embodiments are applicable to allsuch systems without limitation; including, embodiments that minimizenuclease re-cleavage, embodiments for making SNPs with precision at anintended residue, embodiments for making indels with precision at anintended residue and placement of the allele that is being introgressedat the DNA-binding site.

Gene Editing to Avoid Re-Binding by Nuclease Systems

Experimental results suggested that targeted (endo)nuclease systems wereeffectively cutting dsDNA at the intended cognate sites. Analysis of thedata suggested that the nucleases would bind to sites that had alreadybeen templated and re-cleave the site, causing a reversion of the dsDNAto its original sequence. Targeted nuclease systems include a motif thatbinds to the cognate DNA, either by protein-to-DNA binding, or bynucleic acid-to-DNA binding. Experiments demonstrated that templatesthat contain polymorphisms can be selected to confound the re-binding orre-cutting by the targeted nuclease, thereby increasing significantlythe number of precisely introgressed cellular clones.

Embodiments for reducing re-binding include a method ofhomology-directed repair (HDR) to introgress an exogenous allele intochromosomal DNA of a cell, comprising introducing a targeted nucleasesystem and a HDR template that comprises the exogenous allele into thecell, with the targeted nuclease system comprising a DNA-binding memberfor specifically binding an endogenous cognate sequence in thechromosomal DNA, wherein the targeted nuclease system and the HDRtemplate operate to alter the chromosomal DNA to have identity to theHDR template sequence and to introgress the exogenous allele into thechromosomal DNA in place of an endogenous allele. In one embodiment theHDR template sequence is designed to reduce specific binding of theDNA-binding member to the HDR template sequence. In one embodiment theHDR template sequence is designed to introduce a polymorphism intendedto reduce the specific binding of the DNA-binding member to genomicsequence once introgressed. Alternatively, the DNA-binding member of thetargeted nuclease can be designed to recognize nucleotide sequences thataren't present in endogenous or exogenous sequence. Whereas the level ofthis hobbled DNA-binding member is sufficient to enable cleavage of theendogenous allele, the intended polymorphisms from the HDR templatefurther alter the target site and decreases re-cleavage of preciselyintrogressed alleles. This results in a higher frequency of cellularclones within a population that contain those precise introgressionevents.

The term allele means one of two or more forms of a gene. A populationor species of organisms typically includes multiple alleles at eachlocus among various individuals. Allelic variation at a locus ismeasurable as the number of alleles (polymorphisms) present, or theproportion of heterozygotes in the population. The term natural alleleas used herein means an allele found in nature in the same species oforganism that is being modified. The term novel allele means anon-natural allele. A human allele placed into a goat is a novel allele.The term synthetic allele means an allele that has not yet been found innature. An exogenous allele is one that is introduced into an organism,and the endogenous allele is the one that is already in the cell,usually the one that is in the organism in its wild-type unmodifiedstate. Animals that are heterozygous have two alleles. In some cases, itis desirable to introduce an exogenous allele to make an animalhomozygous for an allele that is already present in the heterozygousanimal. Movement of an allele interspecies means from one species ofanimal to another and intraspecies means movement between animals of thesame species. The term exogenous allele is broad and includes DNA with,e.g., native, novel or synthetic SNPs or indels, reporters, endonucleasedigestion sites, promoters, and vectors.

Homology directed repair (HDR) is a mechanism in cells to repair ssDNAand double stranded DNA (dsDNA) lesions. This repair mechanism can beused by the cell when there is an HDR template present that has asequence with significant homology to the lesion site. Specific binding,as that term is commonly used in the biological arts, refers to amolecule that binds to a target with a relatively high affinity comparedto non-target sequences, and generally involves a plurality ofnon-covalent interactions, such as electrostatic interactions, van derWaals interactions, hydrogen bonding, and the like. Specific bindinginvolves processes of binding to a substrate and releasing from asubstrate; as such it can be affected by changes in the efficiency ofbinding and release from a substrate as well as by a strength of thebinding to the substrate. Accordingly, a reduction in specific bindingmay result from a lesser affinity to a substrate that reduces the numberof binding events, or it may result from a reduced strength of bindingto the substrate that reduces how long the binding is maintained. In thecontext of targeted endonucleases, without being bound to a particulartheory, a change in specific binding of the endonuclease or guidesequence to the DNA can affect not only that actual binding but also beinvolved in an incompletely understood process of forming complexes withtargeted and/or template DNA or RNA. Therefore specific binding can bemeasured relative to the actual DNA-binding events and is a usefulfeature for manipulating those processes, even if the actual events atthe chromosomal level involve more or less than actual DNA-binding.Specific hybridization is a form of specific binding between nucleicacids that have complementary sequences. Proteins can also specificallybind to DNA, for instance, in TALENs or CRISPR/Cas9 systems or by Gal4motifs. Introgression of an allele refers to a process of copying anexogenous allele over an endogenous allele with a template-guidedprocess. The endogenous allele might actually be excised and replaced byan exogenous nucleic acid allele in some situations but present theoryis that the process is a copying mechanism. Since alleles are genepairs, there is significant homology between them. The allele might be agene that encodes a protein, or it could have other functions such asencoding a bioactive RNA chain or providing a site for receiving aregulatory protein or RNA.

The HDR template is a nucleic acid that comprises a sequence that, wheninserted into the target genome, results in an altered allele. Thetemplate may be a dsDNA or a single-stranded DNA (ssDNA). ssDNAtemplates are preferably from about 20 to about 5000 residues althoughother lengths can be used. Artisans will immediately appreciate that allranges and values within the explicitly stated range are contemplated;e.g., from 500 to 1500 residues, from 20 to 100 residues, and so forth.The template may further comprise flanking sequences that providehomology to DNA adjacent to the endogenous allele. The template may alsocomprise a sequence that is bound to a targeted nuclease system, and isthus the cognate binding site for the system's DNA-binding member. Theterm cognate refers to two biomolecules that typically interact, forexample, a receptor and its ligand. In the context of HDR processes, oneof the biomolecules may be designed with a sequence to bind with anintended, i.e., cognate, DNA site or protein site.

One embodiment for reducing specific binding to a targeted nucleasesystem comprises making changes in the HDR template relative to itsalignment with the endogenous DNA. One type of change is designed tocreate mismatches between the cognate members. One change is aninsertion or a deletion of one or more residues. Another change is asubstitution of one residue for another residue that does not promotebinding. The term residue refers to a unit in a molecular chain, e.g.,an amino acid in a protein or a base in a nucleic acid. One place tomake the change is at the cognate binding site for the system'sDNA-binding member.

Another type of change is designed to interfere with operation of thenucleases by making the change is in the spacer in systems that operatewith a spacer, e.g., TALENs pairs, the change may be made in the spacerarea. These changes may include a deletion, e.g., so that the nucleasesare hindered from making cuts. These various changes are generallyreferred to as mismatches herein since they create mismatches when thesequences are aligned; in this context, a deletion, insertion, orsubstitution is a mismatch. Artisans routinely make alignments ofsequences so that mismatches are readily identified with specificity.Pairs of nucleases require a spacing that provides a cooperativity;their activity can be disrupted by additions or subtractions to thespacer.

Further embodiments place a mismatch in the exogenous allele. Thesystem's DNA-binding member is designed to bind at a site that at leastpartially overlaps with the endogenous allele. Once it is introgressedto have identity with the exogenous allele, the DNA-binding member hasreduced binding. The DNA-binding member's cognate site thus changes froma preferred endogenous allele to a not-preferred exogenous allele. Thecognate site may encompass all of the allele, or just a part of it. Itis surprising that the introduction of a mismatch into the exogenousallele is required to stabilize the introgression of the exogenousallele. Apparently the problem of re-cleavage has a very large impact onstability of introgressed alleles. The data that shows this impact wasnot previously obtained by others because processes with a comparableefficiency were not conventionally available.

Embodiments include creating, with an HDR templating process, mismatchesat these various places by insertion, deletion, or substitution of aresidue. For instance, from 1-60 residues may be inserted, deleted, orsubstituted; artisans will immediately appreciate that all ranges andvalues within the explicitly stated range are contemplated; e.g., 1-3residues, at least 10 residues, 4 residues, 4-20 residues, and so forth.One or more of these may be combined, e.g., an insertion at one place, adeletion at another, and a substitution at other places.

Embodiments include designing the DNA-binding member of the targetingendonuclease to place a mismatch in the DNA-binding member sequence asaligned with the endogenous chromosomal DNA. The mismatch wouldtypically also be a mismatch for the exogenous DNA. These mismatchesreduce targeted nuclease rebinding. Further mismatches may be used incombination with this method as already described, e.g., with theDNA-binding sites of the endonucleases chosen at positions whereinintrogression of the exogenous allele; the HDR template havingmismatches at the DNA-binding cognates; or in the spacer region tochange the spacing.

These various embodiments can be performed in a reporter-free system andto make an SNP or an embodiment relating to an SNP. The cells or animalsmay be, e.g., vertebrate, livestock, primate, swine, cow, horse, sheep,goat, chicken, rabbit, fish, dog, mouse, cat, rat, and laboratoryanimal.

Zinc Finger Nucleases

Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to target uniquesequences within complex genomes. By taking advantage of endogenous DNArepair machinery, these reagents can be used to alter the genomes ofhigher organisms. ZFNs may be used in methods for inactivating genes.

A zinc finger DNA-binding domain has about 30 amino acids and folds intoa stable structure. Each finger primarily binds to a triplet within theDNA substrate. Amino acid residues at key positions contribute to mostof the sequence-specific interactions with the DNA site. These aminoacids can be changed while maintaining the remaining amino acids topreserve the necessary structure. Binding to longer DNA sequences isachieved by linking several domains in tandem. Other functionalitieslike non-specific FokI cleavage domain (N), transcription activatordomains (A), transcription repressor domains (R) and methylases (M) canbe fused to a ZFPs to form ZFNs respectively, zinc finger transcriptionactivators (ZFA), zinc finger transcription repressors (ZFR, and zincfinger methylases (ZFM). Materials and methods for using zinc fingersand zinc finger nucleases for making genetically modified animals aredisclosed in, e.g., U.S. Pat. No. 8,106,255 US20120192298,US20110023159, and US20110281306.

TALENs

The term TALEN, as used herein, is broad and includes a monomeric TALENthat can cleave double stranded DNA without assistance from anotherTALEN, e.g., as in Beurdeley, M. et al. Compact designer TALENs forefficient genome engineering. Nat. Commun. 4:1762 doi:10.1038/ncomms2782 (2013). The term TALEN is also used to refer to oneor both members of a pair of TALENs that are engineered to work togetherto cleave DNA at the same site. TALENs that work together may bereferred to as a left-TALEN and a right-TALEN, which references thehandedness of DNA or a TALEN-pair.

One of the challenges to making TALEN-modified livestock or otheranimals is that the efficiency of making a modification to an animalcell is only a few percent with conventional best practices. Achievementof a deletion or an insertion at an intended site does not necessarilymean success because it may not actually create the intended effect,such as expressing an exogenous protein or stopping expression of anendogenous protein. Even a low efficiency can be useful for the creationof genetically modified lower animals such as fruit flies or micebecause they have short and prolific reproductive cycles that providefor the creating, testing, and screening of hundreds of animals todetermine if there are a few that have been successfully modified. Theselevels of efficiency that are conventionally achieved, however, are notsuited to livestock artiodactyls that have much longer gestational timesand comparatively few progeny per pregnancy. U.S. Ser. No. 13/404,662filed Feb. 24, 2012 “Genetically modified animals and methods for makingthe same”, which is hereby incorporated herein by reference for allpurposes (in case of conflict, the specification is controlling)provides certain methods that address these conventional limitations.

Another barrier to using TALENs to modify livestock is thatTALEN-mediated modification of DNA in primary cells is difficult becausethe cells are unstable. U.S. Pub. No. 2011/0197290 filed Feb. 11, 2011provides useful methods for modifying these cells, and is herebyincorporated herein by reference for all purposes; in case of conflict,the specification is controlling. Indeed, it is shown herein thatfrequency of TALEN-modified cells decreases significantly over time inthe absence of enrichment or selection methods. Without being bound to aparticular theory, it is theorized that DNA cleavage at non-intendedsites can compromise the stability of the cell by inducing apoptosis ordisabling non-target genes.

The term primary cell means a cell isolated from a living animal,wherein the cell has undergone between 0 and 2, 0 and 3, 0 and 4, 0 and5, 0 and 6, 0 and 7, 0 and 8, 0 and 9, or 0 and 10 replications sinceits isolation from the tissue. TALENs may be used to make geneticallymodified artiodactyl primary cells. These modifications are suited tomaking founders of genetically modified animal lines by cloning. Alsodescribed herein are direct-embryonic injections that that may be usedto modify zygotes or embryos, with the modified zygotes or embryos beingsuited to implant into surrogate females for gestation and delivery offounder animal lines.

As a result, techniques customarily used to create and test transformedcells for successful genetic modification can not be used in primarycells due to their propensity to senesce. TALEN-modified cells arecustomarily destroyed to assay their genetic modification, or isolatedto grow clonal lines with many identical cells from one parent. However,primary cells are inherently unstable and typically undergo geneticchanges, senescence, and/or cell death when attempts are made togenetically modify and clonally expand them. TALEN-modified cells areeven less stable, as documented herein for the first time. As a result,it is unreasonable to expect high rates of success when usingconventional approaches that involve modifying a primary cell forsomatic cell nuclear transfer or other animal cloning technique. Asreported herein, however, TALENs have been used to make geneticallymodified artiodactyl primary cells. These modifications are suited tomaking founders of genetically modified animal lines by cloning ordirect-embryonic injections. Also described herein are direct-embryonicinjections that were used to modify zygotes, with the modified zygotesbeing suited to implant into surrogate females for gestation anddelivery of founder animal lines.

A typical approach to testing for an actual TALEN-mediatedinsertion/deletion event is to sequence the modified cell or zygote,which is a destructive process. Thus when a zygote or embryo is modifiedbefore implantation to a surrogate, its modification cannot be verifiedwith any degree of convenience until the animal is born. It is notconventionally appreciated that an actual production process for makinggenetically modified animals by cloning will benefit from a process fortesting for the presence of a genetic modification. There are inventionspresented herein that provide for an indication of genetic modificationat the single cell, zygote, or oocyte stage. As shown herein, expressionof a reporter gene that is not coupled to TALEN modification is, despitenot being part of the reporter gene expression cassette, nonethelessgenerally predictive of a desired genetic modification. Morespecifically, the expression of the reporter gene indicates that thenucleic acids were effectively delivered and being expressed in a cellor embryo; a reporter-expressing cell or embryo is more likely to haveundergone TALEN-based modification.

Another technique for making modified organisms was the use of aco-transfection, co-selection technique. The cells that express thereporter are selected for, and may be used for making geneticallymodified animals. The reporter may be chosen to require transposaseactivity. Without being bound to a specific theory, it is theorized thatcells that have undergone transposition have 1) been transfected and 2)been competent for double stranded DNA repair, thus increasing thelikelihood of TALEN-based modification in selected clones. This alsofacilitates enrichment/selection for transposed cells (and by extensionTALEN-modified cells). The fact that the transposon is operably but notphysically linked to the TALEN modification permits their segregationaway from each other by breeding. A benefit of a co-transfectionstrategy is that the reporter, or reporters, may be placed on achromosome that is not the same chromosome that is modified by TALENs.This process provides for the creation of founder animals that have noreporter genes. For example, some animals were made by using plasmidscarrying reporter genes that were independent of the geneticmodification, which was orchestrated separately in the cells. Thisscheme was based on a theory of operation that cells that incorporatenew reporter genes will also incorporate genetic modifications. Forinstance, data provided herein shows that cells can be transfected withfour independent plasmids and the successful incorporation of the geneproduct of one plasmid is predictive of successful incorporation of theother plasmid gene products and also for the success of TALEN-mediatedchanges. Conventional wisdom is that transfection with so many plasmidswould not be successful and would yield unhealthy cells. Unexpectedly,however, these techniques were effective.

Miller et al. (Miller et al. (2011) Nature Biotechnol 29:143) reportedmaking TALENs for site-specific nuclease architecture by linking TALtruncation variants to the catalytic domain of FokI nuclease. Theresulting TALENs were shown to induce gene modification in immortalizedhuman cells by means of the two major eukaryotic DNA repair pathways,non-homologous end joining (NHEJ) and homology directed repair. TheTALENs can be engineered for specific binding. Specific binding, as thatterm is commonly used in the biological arts, refers to a molecule thatbinds to a target with a relatively high affinity compared to non-targettissues, and generally involves a plurality of non-covalentinteractions, such as electrostatic interactions, van der Waalsinteractions, hydrogen bonding, and the like. Specific bindinginteractions characterize antibody-antigen binding, enzyme-substratebinding, and specifically binding protein-receptor interactions.

The cipher for TALs has been reported (PCT Application WO 2011/072246)wherein each DNA binding repeat is responsible for recognizing one basepair in the target DNA sequence. The residues may be assembled to targeta DNA sequence, with: (a) HD for recognition of C/G; (b) NI forrecognition of A/T; (c) NG for recognition of T/A; (d) NS forrecognition of C/G or A/T or T/A or G/C; (e) NN for 30 recognition ofG/C or A/T; (f) IG for recognition of T/A; (g) N for recognition of C/G;(h) HG for recognition of C/G or T/A; (i) H for recognition of T/A; and(j) NK for recognition of G/C. In brief, a target site for binding of aTALEN is determined and a fusion molecule comprising a nuclease and aseries of RVDs that recognize the target site is created. Upon binding,the nuclease cleaves the DNA so that cellular repair machinery canoperate to make a genetic modification at the cut ends. The term TALENmeans a protein comprising a Transcription Activator-like (TAL) effectorbinding domain and a nuclease domain and includes monomeric TALENs thatare functional per se as well as others that require dimerization withanother monomeric TALEN. The dimerization can result in a homodimericTALEN when both monomeric TALENs are identical or can result in aheterodimeric TALEN when monomeric TALENs are different. TALENs can beused to induce gene modification in immortalized human cells by means ofthe two major eukaryotic DNA repair pathways, non-homologous end joining(NHEJ) and homology directed repair.

Various working examples for the introduction of TALENs into cells orembryos, and the formation of animals therefrom are provided herein.Cells for treatment by TALENs include a cultured cell, an immortalizedcell, a primary cell, a primary somatic cell, a zygote, a germ cell, aprimordial germ cell, a blastocyst, or a stem cell. Example 18 (FIG. 26)details experimental results for modifying spermatogonial stem cells.These cells offer another method for genetic modification of animals,e.g., livestock. Genetic modification or gene edits can be executed invitro in spermatogonial stem cells (male germ-line stem cells, hereinabbreviated GSC's) isolated from donor testes. Modified cells aretransplanted into germ-cell depleted testes of a recipient. Implantedspermatogonial stem cells produce sperm that carry the geneticmodification(s) that can be used for breeding via artificialinsemination (AI) or in vitro fertilization (IVF) to derive founderanimals. This method has advantages beyond generation of geneticallymodified founders. One such advantage is apparent when founders for aparticular disease model are unhealthy and not suitable for growth toreproductive age. The same modifications introduced into GSC's couldthus be implanted into the testes of a healthy individuals allowingpropagation of the line from a healthy animal to generate disease modelsin newborn piglets.

In some embodiments, a monomeric TALEN can be used. TALENs typicallyfunction as dimers across a bipartite recognition site with a spacer,such that two TAL effector domains are each fused to a catalytic domainof the FokI restriction enzyme, the DNA-recognition sites for eachresulting TALEN are separated by a spacer sequence, and binding of eachTALEN monomer to the recognition site allows FokI to dimerize and createa double-strand break within the spacer. Monomeric TALENs also can beconstructed, however, such that single TAL effectors are fused to anuclease that does not require dimerization to function. One suchnuclease, for example, is a single-chain variant of FokI in which thetwo monomers are expressed as a single polypeptide. Other naturallyoccurring or engineered monomeric nucleases also can serve this role.The DNA recognition domain used for a monomeric TALEN can be derivedfrom a naturally occurring TAL effector. Alternatively, the DNArecognition domain can be engineered to recognize a specific DNA target.Engineered single-chain TALENs may be easier to construct and deploy, asthey require only one engineered DNA recognition domain. A dimeric DNAsequence-specific nuclease can be generated using two different DNAbinding domains (e.g., one TAL effector binding domain and one bindingdomain from another type of molecule). TALENs may function as dimersacross a bipartite recognition site with a spacer. This nucleasearchitecture also can be used for target-specific nucleases generatedfrom, for example, one TALEN monomer and one zinc finger nucleasemonomer. In such cases, the DNA recognition sites for the TALEN and zincfinger nuclease monomers can be separated by a spacer of appropriatelength. Binding of the two monomers can allow FokI to dimerize andcreate a double-strand break within the spacer sequence. DNA bindingdomains other than zinc fingers, such as homeodomains, myb repeats orleucine zippers, also can be fused to FokI and serve as a partner with aTALEN monomer to create a functional nuclease.

In some embodiments, a TAL effector can be used to target other proteindomains (e.g., non-nuclease protein domains) to specific nucleotidesequences. For example, a TAL effector can be linked to a protein domainfrom, without limitation, a DNA 20 interacting enzyme (e.g., amethylase, a topoisomerase, an integrase, a transposase, or a ligase), atranscription activators or repressor, or a protein that interacts withor modifies other proteins such as histones. Applications of such TALeffector fusions include, for example, creating or modifying epigeneticregulatory elements, making site-specific insertions, deletions, orrepairs in DNA, controlling gene expression, and modifying chromatinstructure.

The spacer of the target sequence can be selected or varied to modulateTALEN specificity and activity. The flexibility in spacer lengthindicates that spacer length can be chosen to target particularsequences with high specificity. Further, the variation in activity hasbeen observed for different spacer lengths indicating that spacer lengthcan be chosen to achieve a desired level of TALEN activity.

The TALENs described herein as Carlson +63 were surprisingly found to bevery efficient in use. A comparison to the most similar TALENs is shownin FIGS. 16 and 18. Referring to FIG. 16 and using the position numberstherein, there is a leading N-terminal portion from about 1 to about 42,a 5′ portion from about 43 to about 178, and RVD portion from about 179to about 197, a +63 domain from about 198 to about 261, and a FokIportion from about 262 to the end at about 400. A number of residues aredifferent between the sequences, for instance at about 10 positions thatare circled in FIG. 16A. The N-terminal leader portion is alsodifferent, with the Carlson +63 TALEN being about 20 residues shorterand having about 16 other differences. Embodiments of the N-terminalleader portion are sequences of between about 10 to about 30 residues;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated.

FIG. 17 provides a sequence listing for the vector used with the Carlson+63 sequence. Some parts of the vector are indicated in the Figure: theT3 primer binding site, a 5′ UTR, the TALEN 5′ for Carlson +63, aLacZ-stuffer fragment (see Cermak et. al. 2011 for blue white screeningof clones), a Fok I homodimer, a 3′ TALEN+18-+63 (note that 3′TALEN+1-17 provided by last TALEN repeat in final cloning step), a 3′UTR-polyA, and a Poly-C that potentially protects the mRNA fromdegradation. In use, as is known to artisans, the amino acids thatprovide specific binding are inserted in between the portions labeled asthe 5′ portion and the half RVD sequence. FIG. 17 shows the Carlson +63TALEN scaffold with various features for production of mRNA. The vectorhas some features in common with a pT3TS plasmid previously described(Hyatt, T. M. & Ekker, S. C. Vectors and techniques for ectopic geneexpression in zebrafish. Methods Cell Biol 59, 117-126 (1999)). Asignificant improvement to the Hyatt et al. vector was made by removalof a LacZ promoter that was previously located 5′ of the T3 promotersequence indicated in FIG. 17. Removal of the LacZ promoter was found tobe required for reliable cloning of gene specific TALENs and propagationof the plasmid. The Carlson +63 vector has a T3 site for mRNAtranscription with T3 mRNA polymerase. The features include a T3promoter binding site from which transcription can be initiated, 5′ and3′ UTR sequence from the Xenopus β-globin gene, and a poly-C stretch.The 5′ and 3′ portions of the TALEN scaffold flank a LacZ stufferfragment that is removed when the gene specific RVD sequences are clonedin as described in Cermak, T. et al. Efficient design and assembly ofcustom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Research 39, e82 (2011).

Alternative embodiments use alternative mRNA polymerases and cognatebinding sites such as T7 or SP6. Other embodiments relate to the use ofany of several alterations of the UTR sequences; these could benefittranslation of the mRNA. Some examples are: addition of a cytoplasmicpolyadenylation element binding site in the 3′ UTR, or exchanging theXenopus β-globin UTRs with UTR sequences from human, pig, cow, sheep,goat, zebrafish, from genes including B-globin. UTRs from genes may beselected for regulation of expression in embryonic development or incells. Some examples of UTRs that may be useful include β-actin, DEAH(SEQ ID NO: 527), TPT1, ZF42, SKP1, TKT, TP3, DDX5, EIF3A, DDX39, GAPDH,CDK1, Hsp90ab1, Ybx1 f Eif4b Rps27a Stra13, Myc, Paf1 and Foxo1, orCHUK. Such vector or mRNA improvements could be used to direct specialor temporal expression of ectopic TALENs for study of gene depletion atdesired stages of development. TALEN mRNA produced by these vectors aregenerally useful as described herein, including, for example, forcreation of knockout or knockin cells lines or animals to generatemodels of disease, animal improvement or screening of for genes thatinteract with environmental stimuli (example; drugs, heat, cold, UVlight, growth factors, stress).

Embodiments include a vector comprising a sequence having 85% to 100%identity with the Carlson +63 vector or TALEN; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 85%, 90%, and 95%. Embodiments include aCarlson +63 TALEN with a number of conservative substitutions rangingfrom 1 to 50; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated, e.g., 5to 10, 1 to 20, or about 12. Artisans will immediately appreciate thatthe RVD portions of these sequences are to be excluded from thesecomparisons since the RVD sequences are to be changed according to thetarget intended by a user. Embodiments include a TALEN that comprises atleast one portion of a Carlson +63 TALEN chosen from the groupconsisting of N-terminal leader portion, 5′ portion, and +63 domain (and% variations/substitutions thereof).

The Carlson +63 TALEN has a 22-residue N-terminal leader sequence ofMASSPPKKKRKVSWKDASGWSR (SEQ ID NO: 132). Embodiments include a TALENvector or mRNA that comprises at least one portion of a Carlson +63TALEN vector chosen from the group consisting of 3′ primer biding site,5′UTR, lacz stuffer fragment, 3′ TALEN, 3′UTR, PolyC, and nucleic acidsencoding the Carlson +63 N-terminal leader portion, 5′ portion, or +63domain (and variations/substitutions thereof). Alternatively, a sequencemay be assembled using one or more of the alternatives indicated above,e.g., for T7 or SP6 or any of the various alternative UTRs. Embodimentsinclude sequences with between 85% and 100% identity to the same, aswell as a number of conservative substitutions ranging from 0 to 50.

In some embodiments, a monomeric TALEN can be used. TALEN typicallyfunction as dimers across a bipartite recognition site with a spacer,such that two TAL effector domains are each fused to a catalytic domainof the FokI restriction enzyme, the DNA-recognition sites for eachresulting TALEN are separated by a spacer sequence, and binding of eachTALEN monomer to the recognition site allows FokI to dimerize and createa double-strand break within the spacer. Monomeric TALENs also can beconstructed, however, such that single TAL effectors are fused to anuclease that does not require dimerization to function. One suchnuclease, for example, is a single-chain variant of FokI in which thetwo monomers are expressed as a single polypeptide. Other naturallyoccurring or engineered monomeric nucleases also can serve this role.The DNA recognition domain used for a monomeric TALEN can be derivedfrom a naturally occurring TAL effector. Alternatively, the DNArecognition domain can be engineered to recognize a specific DNA target.Engineered single-chain TALENs may be easier to construct and deploy, asthey require only one engineered DNA recognition domain. A dimeric DNAsequence-specific nuclease can be generated using two different DNAbinding domains (e.g., one TAL effector binding domain and one bindingdomain from another type of molecule). TALENs may function as dimersacross a bipartite recognition site with a spacer. This nucleasearchitecture also can be used for target-specific nucleases generatedfrom, for example, one TALEN monomer and one zinc finger nucleasemonomer. In such cases, the DNA recognition sites for the TALEN and zincfinger nuclease monomers can be separated by a spacer of appropriatelength. Binding of the two monomers can allow FokI to dimerize andcreate a double-strand break within the spacer sequence. DNA bindingdomains other than zinc fingers, such as homeodomains, myb repeats orleucine zippers, also can be fused to FokI and serve as a partner with aTALEN monomer to create a functional nuclease.

The term nuclease includes exonucleases and endonucleases. The termendonuclease refers to any wild-type or variant enzyme capable ofcatalyzing the hydrolysis (cleavage) of bonds between nucleic acidswithin a DNA or RNA molecule, preferably a DNA molecule. Non-limitingexamples of endonucleases include type II restriction endonucleases suchas FokI, HhaI, HindIII, NotI, BbvCI, EcoRI, BgIII, and AlwI.Endonucleases comprise also rare-cutting endonucleases when havingtypically a polynucleotide recognition site of about 12-45 basepairs(bp) in length, more preferably of 14-45 bp. Rare-cutting endonucleasesinduce DNA double-strand breaks (DSBs) at a defined locus. Rare-cuttingendonucleases can for example be a homing endonuclease, a chimericZinc-Finger nuclease (ZFN) resulting from the fusion of engineeredzinc-finger domains with the catalytic domain of a restriction enzymesuch as FokI or a chemical endonuclease. In chemical endonucleases, achemical or peptidic cleaver is conjugated either to a polymer ofnucleic acids or to another DNA recognizing a specific target sequence,thereby targeting the cleavage activity to a specific sequence. Chemicalendonucleases also encompass synthetic nucleases like conjugates oforthophenanthroline, a DNA cleaving molecule, and triplex-formingoligonucleotides (TFOs), known to bind specific DNA sequences. Suchchemical endonucleases are comprised in the term “endonuclease”according to the present invention. Examples of such endonucleaseinclude I-See I, I-Chu I, I-Cre I, I-Csm I, PI-See L PI-Tti L PI-Mtu I,I-Ceu I, I-See IL I-See III, HO, PI-Civ I, PI-Ctr L PI-Aae I, PI-BsuPI-Dha I, PI-Dra L PI May L PI-Meh I, PI-Mfu L PI-Mfl I, PI-Mga L PI-MgoI, PI-Min L PI-Mka L PI-Mle I, PI-Mma I, PI-30 Msh L PI-Msm I, PI-Mth I,PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu L PI-Rma I, PI-Spb I, PI-Ssp LPI-Fae L PI-Mja I, PI-Pho L PI-Tag L PI-Thy I, PI-Tko I, PI-Tsp I,I-MsoI.

A genetic modification made by TALENs or other tools may be, forexample, chosen from the list consisting of an insertion, a deletion,insertion of an exogenous nucleic acid fragment, and a substitution. Theterm “insertion” is used broadly to mean either literal insertion intothe chromosome or use of the exogenous sequence as a template forrepair. In general, a target DNA site is identified and a TALEN-pair iscreated that will specifically bind to the site. The TALEN is deliveredto the cell or embryo, e.g., as a protein, mRNA or by a vector thatencodes the TALEN. The TALEN cleaves the DNA to make a double-strandbreak that is then repaired, often resulting in the creation of anindel, or incorporating sequences or polymorphisms contained in anaccompanying exogenous nucleic acid that is either inserted into thechromosome or serves as a template for repair of the break with amodified sequence. This template-driven repair is a useful process forchanging a chromosome, and provides for effective changes to cellularchromosomes.

The term exogenous nucleic acid means a nucleic acid that is added tothe cell or embryo, regardless of whether the nucleic acid is the sameor distinct from nucleic acid sequences naturally in the cell. In somecases, the exogenous nucleic acid differs in sequence from any nucleicacid sequence that occurs naturally within the cell. The term nucleicacid fragment is broad and includes a chromosome, expression cassette,gene, DNA, RNA, mRNA, or portion thereof. The cell or embryo may be, forinstance, chosen from the group consisting of livestock, an artiodactyl,cattle, a swine, a sheep, a goat, a chicken, a rabbit, and a fish. Theterm “livestock” means domesticated animals that are raised ascommodities for food or biological material. The term artiodactyl meansa hoofed mammal of the order Artiodactyla, which includes cattle, deer,camels, hippopotamuses, sheep, and goats, that have an even number oftoes, usually two or sometimes four, on each foot.

Some embodiments involve a composition or a method of making agenetically modified livestock and/or artiodactyl comprising introducinga TALEN-pair into livestock and/or an artiodactyl cell or embryo thatmakes a genetic modification to DNA of the cell or embryo at a site thatis specifically bound by the TALEN-pair, and producing the livestockanimal/artiodactyl from the cell. Direct injection may be used for thecell or embryo, e.g., into a zygote, blastocyst, or embryo.Alternatively, the TALEN and/or other factors may be introduced into acell using any of many known techniques for introduction of proteins,RNA, mRNA, DNA, or vectors. Genetically modified animals may be madefrom the embryos or cells according to known processes, e.g.,implantation of the embryo into a gestational host, or various cloningmethods. The phrase “a genetic modification to DNA of the cell at a sitethat is specifically bound by the TALEN”, or the like, means that thegenetic modification is made at the site cut by the nuclease on theTALEN when the TALEN is specifically bound to its target site. Thenuclease does not cut exactly where the TALEN-pair binds, but rather ata defined site between the two binding sites.

Some embodiments involve a composition or a treatment of a cell that isused for cloning the animal. The cell may be a livestock and/orartiodactyl cell, a cultured cell, a primary cell, a primary somaticcell, a zygote, a germ cell, a primordial germ cell, or a stem cell. Forexample, an embodiment is a composition or a method of creating agenetic modification comprising exposing a plurality of primary cells ina culture to TALEN proteins or a nucleic acid encoding a TALEN orTALENs. The TALENs may be introduced as proteins or as nucleic acidfragments, e.g., encoded by mRNA or a DNA sequence in a vector.

Genetic modification of cells may also include insertion of a reporter.The reporter may be, e.g., a florescent marker, e.g., green fluorescentprotein and yellow fluorescent protein. The reporter may be a selectionmarker, e.g., puromycin, ganciclovir, adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolatereductase (DHFR), hygromycin-B-phosphtransferase, thymidine kinase (TK),or xanthin-guanine phosphoribosyltransferase (XGPRT). Vectors for thereporter, selection marker, and/or one or more TALEN may be a plasmid,transposon, transposase, viral, or other vectors, e.g., as detailedherein.

TALENs may be directed to a plurality of DNA sites. The sites may beseparated by several thousand or many thousands of base pairs. The DNAcan be rejoined by cellular machinery to thereby cause the deletion ofthe entire region between the sites. Embodiments include, for example,sites separated by a distance between 1-5 megabases or between 50% and80% of a chromosome, or between about 100 and about 1,000,000 basepairs;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., from about1,000 to about 10,000 basepairs or from about 500 to about 500,000basepairs. Alternatively, exogenous DNA may be added to the cell orembryo for insertion of the exogenous DNA, or template-driven repair ofthe DNA between the sites. Modification at a plurality of sites may beused to make genetically modified cells, embryos, artiodactyls, andlivestock. One or more genes may be chosen for complete or at leastpartial deletion, including a sexual maturation gene or a cis-actingfactor thereof.

The possibility and efficiency of generating TALEN-mediated indels inspermatogonial stem cells was explored by transfection of plasmidsencoding TALENs targeted to exon 7 of the porcine Duchene MuscularDystrophy locus (DMD). Testing of several nuclefection conditions,plasmid quantities and incubation temperature yielded a maximumefficiency of 19% NHEJ despite a germ cell transfection rate of 25%, asshown in FIG. 26. TALEN activity was highest in replicates cultured at30° C. GSCs remained viable after over 5 days of culture at 30° C.,though overall, germ cell survival was higher at 37° C. Transfection ofTALEN encoding mRNA, versus plasmid DNA, resulted in both greateractivity and viability of livestock somatic cells and GSCs. Notably,while peak activity of mRNA transfection did not exceed plasmid DNAtransfection in this experiment, a significantly lower quantity of mRNAwas required to achieve the same level of modification. Example 19details successful TALEN-stimulated HDR in primordial germ cells(avian).

In some embodiments, a monomeric TALEN can be used. TALEN typicallyfunction as dimers across a bipartite recognition site with a spacer,such that two TAL effector domains are each fused to a catalytic domainof the FokI restriction enzyme, the DNA-recognition sites for eachresulting TALEN are separated by a spacer sequence, and binding of eachTALEN monomer to the recognition site allows FokI to dimerize and createa double-strand break within the spacer. Monomeric TALENs also can beconstructed, however, such that single TAL effectors are fused to anuclease that does not require dimerization to function. One suchnuclease, for example, is a single-chain variant of FokI in which thetwo monomers are expressed as a single polypeptide. Other naturallyoccurring or engineered monomeric nucleases also can serve this role.The DNA recognition domain used for a monomeric TALEN can be derivedfrom a naturally occurring TAL effector. Alternatively, the DNArecognition domain can be engineered to recognize a specific DNA target.Engineered single-chain TALENs may be easier to construct and deploy, asthey require only one engineered DNA recognition domain. A dimeric DNAsequence-specific nuclease can be generated using two different DNAbinding domains (e.g., one TAL effector binding domain and one bindingdomain from another type of molecule). TALENs may function as dimersacross a bipartite recognition site with a spacer. This nucleasearchitecture also can be used for target-specific nucleases generatedfrom, for example, one TALEN monomer and one zinc finger nucleasemonomer. In such cases, the DNA recognition sites for the TALEN and zincfinger nuclease monomers can be separated by a spacer of appropriatelength. Binding of the two monomers can allow FokI to dimerize andcreate a double-strand break within the spacer sequence. DNA bindingdomains other than zinc fingers, such as homeodomains, myb repeats orleucine zippers, also can be fused to FokI and serve as a partner with aTALEN monomer to create a functional nuclease.

Recombinases

Embodiments of the invention include administration of a TALEN or TALENswith a recombinase or other DNA-binding protein associated with DNArecombination. A recombinase forms a filament with a nucleic acidfragment and, in effect, searches cellular DNA to find a DNA sequencesubstantially homologous to the sequence. An embodiment of aTALEN-recombinase embodiment comprises combining a recombinase with anucleic acid sequence that serves as a template for HDR. The HDRtemplate sequence has substantial homology to a site that is targetedfor cutting by the TALEN/TALEN pair. As described herein, the HDRtemplate provides for a change to the native DNA, by placement of anallele, creation of an indel, insertion of exogenous DNA, or with otherchanges. The TALEN is placed in the cell or embryo by methods describedherein as a protein, mRNA, or by use of a vector. The recombinase iscombined with the HDR template to form a filament and placed into thecell. The recombinase and/or HDR template that combines with therecombinase may be placed in the cell or embryo as a protein, an mRNA,or with a vector that encodes the recombinase. The disclosure of US Pub2011/0059160 (U.S. Ser. No. 12/869,232) is hereby incorporated herein byreference for all purposes; in case of conflict, the specification iscontrolling. The term recombinase refers to a genetic recombinationenzyme that enzymatically catalyzes, in a cell, the joining ofrelatively short pieces of DNA between two relatively longer DNAstrands. Recombinases include Cre recombinase, Hin recombinase, RecA,RAD51, Cre, and FLP. Cre recombinase is a Type I topoisomerase from P1bacteriophage that catalyzes site-specific recombination of DNA betweenloxP sites. Hin recombinase is a 21 kD protein composed of 198 aminoacids that is found in the bacteria Salmonella. Hin belongs to theserine recombinase family of DNA invertases in which it relies on theactive site serine to initiate DNA cleavage and recombination. RAD51 isa human gene. The protein encoded by this gene is a member of the RAD51protein family which assist in repair of DNA double strand breaks. RAD51family members are homologous to the bacterial RecA and yeast Rad51genes. Cre recombinase is an enzyme that is used in experiments todelete specific sequences that are flanked by loxP sites. FLP refers toFlippase recombination enzyme (FLP or Flp) derived from the 2μ plasmidof the baker's yeast Saccharomyces cerevisiae.

Herein, “RecA” or “RecA protein” refers to a family of RecA-likerecombination proteins having essentially all or most of the samefunctions, particularly: (i) the ability to position properlyoligonucleotides or polynucleotides on their homologous targets forsubsequent extension by DNA polymerases; (ii) the ability topologicallyto prepare duplex nucleic acid for DNA synthesis; and, (iii) the abilityof RecA/oligonucleotide or RecA/polynucleotide complexes efficiently tofind and bind to complementary sequences. The best characterized RecAprotein is from E. coli; in addition to the original allelic form of theprotein a number of mutant RecA-like proteins have been identified, forexample, RecA803. Further, many organisms have RecA-like strand-transferproteins including, for example, yeast, Drosophila, mammals includinghumans, and plants. These proteins include, for example, Rec1, Rec2,Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2 and DMC1. An embodiment ofthe recombination protein is the RecA protein of E. coli. Alternatively,the RecA protein can be the mutant RecA-803 protein of E. coli, a RecAprotein from another bacterial source or a homologous recombinationprotein from another organism.

RecA is known for its recombinase activity to catalyze strand exchangeduring the repair of double-strand breaks by homologous recombination(McGrew and Knight, 2003) Radding, et al., 1981; Seitz et al., 1998).RecA has also been shown to catalyze proteolysis, e.g., of the LexA andλ repressor proteins, and to possess DNA-dependent ATPase activity.After a double-strand break occurs from ionizing radiation or some otherinsult, exonucleases chew back the DNA ends 5′ to 3′, thereby exposingone strand of the DNA (Cox, 1999; McGrew and Knight, 2003). Thesingle-stranded DNA becomes stabilized by single-strand binding protein(SSB). After binding of SSB, RecA binds the single-stranded (ss) DNA andforms a helical nucleoprotein filament (referred to as a filament or apresynaptic filament). During DNA repair, the homology-searchingfunctions of RecA direct the filament to homologous DNA and catalyzehomologous base pairing and strand exchange. This results in theformation of DNA heteroduplex. After strand invasion, DNA polymeraseelongates the ssDNA based on the homologous DNA template to repair theDNA break, and crossover structures or Holliday junctions are formed.RecA also shows a motor function that participates in the migration ofthe crossover structures (Campbell and Davis, 1999).

Recombinase activity comprises a number of different functions. Forexample, polypeptide sequences having recombinase activity are able tobind in a non-sequence-specific fashion to single-stranded DNA to form anucleoprotein filament. Such recombinase-bound nucleoprotein filamentsare able to interact in a non-sequence-specific manner with adouble-stranded DNA molecule, search for sequences in thedouble-stranded molecule that are homologous to sequences in thefilament, and, when such sequences are found, displace one of thestrands of the double-stranded molecule to allow base-pairing betweensequences in the filament and complementary sequences in one of thestrands of the double stranded molecule. Such steps are collectivelydenoted “synapsis.”

RecA and RecA-like proteins (called Rad51 in many eukaryotic species)have been examined for stimulating gene targeting and homologousrecombination in a variety of eukaryotic systems. In tobacco cells,expression of bacterial RecA containing a nuclear localization signal(NLS) increases the repair of mitomycin C-induced DNA damage byhomologous recombination and somatic intrachromosomal recombination(recombination between homologous chromosomes) from three to ten fold(Reiss et al., 1996). Expression of NLSRecA in tobacco can alsostimulate sister chromatid exchange 2.4-fold over wild-type levels(Reiss et al., 2000). In somatic mammalian cells, overexpression ofNLSRecA stimulates gene-targeting by homologous recombination 10-fold(Shcherbakova et al., 2000). However, in human cells, overexpression ofa human homologue of RecA, hRAD51, only stimulates recombination 2 to3-fold over wild type levels under the antibiotic selection (Yanez andPorter, 1999). In zebrafish, a mutant form of the enhanced greenfluorescent protein (EGFP) was corrected at low frequency by injectingssDNA-RecA filaments directly (Cui et al., 2003). Rad52, a member of theRad51 epistasis group, also promotes single-strand annealing and lowlevel gene disruption in zebrafish using mutated oligonucleotides(Takahashi and Dawid, 2005). Taken together, these studies indicate thatectopic expression of RecA or Rad51 results in a modest stimulation ofhomologous recombination but does not increase levels sufficiently to beuseful for gene-targeting.

Thus recombinase activities include, but are not limited to,single-stranded DNA-binding, synapsis, homology searching, duplexinvasion by single-stranded DNA, heteroduplex formation, ATP hydrolysisand proteolysis. The prototypical recombinase is the RecA protein fromE. coli. See, for example, U.S. Pat. No. 4,888,274. ProkaryoticRecA-like proteins have also been described in Salmonella, Bacillus andProteus species. A thermostable RecA protein, from Thermus aquaticus,has been described in U.S. Pat. No. 5,510,473. A bacteriophage T4homologue of RecA, the UvsX protein, has been described. RecA mutants,having altered recombinase activities, have been described, for example,in U.S. Pat. Nos. 6,774,213; 7,176,007 and 7,294,494. Plant RecAhomologues are described in, for example, U.S. Pat. Nos. 5,674,992;6,388,169 and 6,809,183. RecA fragments containing recombinase activityhave been described, for example, in U.S. Pat. No. 5,731,411. MutantRecA proteins having enhanced recombinase activity such as, for example,RecA803 have been described. See, for example, Madiraju et al. (1988)Proc. Natl. Acad. Sci. USA 85:6592-6596.

A eukaryotic homologue of RecA, also possessing recombinase activity, isthe Rad51 protein, first identified in the yeast Saccharomycescerevisiae. See Bishop et al., (1992) Cell 69:439-56; Shinohara et al,(1992) Cell: 457-70; Aboussekhra, et al., (1992) Mol. Cell. Biol. 72,3224-3234 and Basile et al., (1992) Mol. Cell. Biol. 12, 3235-3246.Plant Rad51 sequences are described in U.S. Pat. Nos. 6,541,684;6,720,478; 6,905,857 and 7,034,117. Another yeast protein that ishomologous to RecA is the Dmc1 protein. RecA/Rad51 homologues inorganisms other than E. coli and S. cerevisiae have been described.Morita et al. (1993) Proc. Natl. Acad. Sci. USA 90:6577-6580; Shinoharaet al. (1993) Nature Genet. 4:239-243; Heyer (1994) Experientia50:223-233; Maeshima et al. (1995) Gene 160:195-200; U.S. Pat. Nos.6,541,684 and 6,905,857.

Herein, “RecA” or “RecA protein” refers to a family of RecA-likerecombination proteins having essentially all or most of the samefunctions, particularly: (i) the ability to position properlyoligonucleotides or polynucleotides on their homologous targets forsubsequent extension by DNA polymerases; (ii) the ability topologicallyto prepare duplex nucleic acid for DNA synthesis; and, (iii) the abilityof RecA/oligonucleotide or RecA/polynucleotide complexes efficiently tofind and bind to complementary sequences. The best characterized RecAprotein is from E. coli; in addition to the original allelic form of theprotein a number of mutant RecA-like proteins have been identified, forexample, RecA803. Further, many organisms have RecA-like strand-transferproteins including, for example, yeast, Drosophila, mammals includinghumans, and plants. These proteins include, for example, Rec1, Rec2,Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2 and DMC1. An embodiment ofthe recombination protein is the RecA protein of E. coli. Alternatively,the RecA protein can be the mutant RecA-803 protein of E. coli, a RecAprotein from another bacterial source or a homologous recombinationprotein from another organism.

Additional descriptions of proteins having recombinase activity arefound, for example, in Fugisawa et al. (1985) Nucl. Acids Res. 13:7473;Hsieh et al. (1986) Cell 44:885; Hsieh et al. (1989) J. Biol. Chem.264:5089; Fishel et al. (1988) Proc. Natl. Acad. Sci. USA 85:3683;Cassuto et al. (1987) Mol. Gen. Genet. 208:10; Ganea et al. (1987) Mol.Cell Biol. 7:3124; Moore et al. (1990) J. Biol. Chem.:11108; Keene etal. (1984) Nucl. Acids Res. 12:3057; Kimiec (1984) Cold Spring HarborSymp. 48:675; Kimeic (1986) Cell 44:545; Kolodner et al. (1987) Proc.Natl. Acad. Sci. USA 84:5560; Sugino et al. (1985) Proc. Natl. Acad,Sci. USA 85: 3683; Halbrook et al. (1989) J. Biol. Chem. 264:21403;Eisen et al. (1988) Proc. Natl. Acad. Sci. USA 85:7481; McCarthy et al.(1988) Proc. Natl. Acad. Sci. USA 85:5854; and Lowenhaupt et al. (1989)J. Biol. Chem. 264:20568, which are incorporated herein by reference.See also Brendel et al. (1997) J. Mol. Evol. 44:528.

Examples of proteins having recombinase activity include recA, recA803,uvsX, and other recA mutants and recA-like recombinases (Roca (1990)Crit. Rev. Biochem. Molec. Biol. 25:415), (Kolodner et al. (1987) Proc.Natl. Acad. Sci. U.S.A. 84:5560; Tishkoff et al. (1991) Molec. Cell.Biol. 11:2593), RuvC (Dunderdale et al. (1991) Nature 354:506), DST2,KEM1 and XRN1 (Dykstra et al. (1991) Molec. Cell. Biol. 11:2583),STPa/DST1 (Clark et al. (1991) Molec. Cell. Biol. 11:2576), HPP-1 (Mooreet al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:9067), other eukaryoticrecombinases (Bishop et al. (1992) Cell 69:439; and Shinohara et al.(1992) Cell 69:457); incorporated herein by reference.

In vitro-evolved proteins having recombinase activity have beendescribed in U.S. Pat. No. 6,686,515. Further publications relating torecombinases include, for example, U.S. Pat. Nos. 7,732,585, 7,361,641,7,144,734. For a review of recombinases, see Cox (2001) Proc. Natl.Acad. Sci. USA 98:8173-8180.

A nucleoprotein filament, or “filament” may be formed. The termfilament, in the context of forming a structure with a recombinase, is aterm known to artisans in these fields. The nucleoprotein filament soformed can then be, e.g., contacted with another nucleic acid orintroduced into a cell. Methods for forming nucleoprotein filaments,wherein the filaments comprise polypeptide sequences having recombinaseactivity and a nucleic acid, are well-known in the art. See, e.g., Cuiet al. (2003) Marine Biotechnol. 5:174-184 and U.S. Pat. Nos. 4,888,274;5,763,240; 5,948,653 and 7,199,281, the disclosures of which areincorporated by reference for the purposes of disclosing exemplarytechniques for binding recombinases to nucleic acids to formnucleoprotein filaments.

In general, a molecule having recombinase activity is contacted with alinear, single-stranded nucleic acid. The linear, single-strandednucleic acid may be a probe. The methods of preparation of such singlestranded nucleic acids are known. The reaction mixture typicallycontains a magnesium ion. Optionally, the reaction mixture is bufferedand optionally also contains ATP, dATP or a nonhydrolyzable ATPanalogue, such as, for example, γ-thio-ATP (ATP-γ-S) or γ-thio-GTP(GTP-γ-S). Reaction mixtures can also optionally contain anATP-generating system. Double-stranded DNA molecules can be denatured(e.g., by heat or alkali) either prior to, or during, filamentformation. Optimization of the molar ratio of recombinase to nucleicacid is within the skill of the art. For example, a series of differentconcentrations of recombinase can be added to a constant amount ofnucleic acid, and filament formation assayed by mobility in an agaroseor acrylamide gel. Because bound protein retards the electrophoreticmobility of a polynucleotide, filament formation is evidenced byretarded mobility of the nucleic acid. Either maximum degree ofretardation, or maximum amount of nucleic acid migrating with a retardedmobility, can be used to indicate optimal recombinase:nucleic acidratios. Protein-DNA association can also be quantitated by measuring theability of a polynucleotide to bind to nitrocellulose.

Creation of Genetically Modified Livestock Via TALEN Technologies;Verification of TALEN Modification; Co-Selection of Modified Cells;Elimination of Reporter Genes from Genetically Modified Animals

TALEN function in livestock embryos was investigated using in vitroprepared (IVP) bovine and porcine embryos. Example 1 describes directinjection of TALENs (a left TALEN and a right TALEN) into bovine embryosto produce genetically modified animals with a modification at the sitewhere the TALENs specifically bound. The modifications includedhomozygous-biallelic and heterozygous-biallelic modifications. TALENmRNAs were directly injected into the embryos and successful geneticmodifications were observed. Expression of the reporter was predictiveof a successful genetic modification, with about 35% of the embryosexpressing the reporter, and about 30% of these animals having aTALEN-based indel. Of the animals with indels, about 35% of them wereeither homozygous or heterozygous bi-allelic mutants (FIG. 4). Directembryo modification using TALENs was thus shown to be a viable approachto livestock genome modification. Embryos may thus be prepared andimplanted into surrogate females for gestation and delivery of animalfounder lines using well known processes. Moreover, it is possible touse a reporter to select cells (e.g., primary cells, zygotes, oocytes,blastocysts) for further use in cloning or other processes.

Methods for TALEN-mediated genetic modification of livestock (orzebrafish, dogs, mice, rats, avian, chicken, or a laboratory animal) bycloning were also developed. Example 2 describes development of suitableTALENs and TALEN modification of somatic primary cells of swine andcows. The efficiency of successful modification was somewhat low and noreporters for measuring success of the modification were used.Nucleofection is a means for introducing foreign nucleic acids into acell with high efficiency, but it is expensive, results in high levelsof cytotoxicity, and is not available to many researchers. Therefore, acommon cationic lipid transfection reagent was used as a vehicle forgenetic modification. As shown in Example 3, despite a less than 5%transfection efficiency with cationic lipids, modification levels weresignificantly enriched by transposon co-selection. Whereas genemodification was below detection in day 3 populations (data not shown)and day 14 populations without transposon-mediated selection,modification levels in selected populations reached 31, 13 and 20percent for DMD7.1, DMD6 and LDLR2.1 respectively (FIG. 7). Transposonco-selection was then applied to cells transfected by nucleofectionwhere >90% transfection efficiency is routine. Transposon co-selectionwas effective for maintenance modified cells transfected byNucleofection, however, with the exception of ACAN12, nucleofection didnot significantly enrich for modified cells over day 3 levels (FIG. 7).Thus, transposon co-selection is an effective enrichment method whentransfection efficiency is low and an effective maintenance method whentransfection efficiency is high. Co-selection processes were alsoeffective when feeder cells were used, as demonstrated in Example 4. Anunexpectedly high proportion of bi-allelic modifications (about 17% toabout 35% depending on the TALEN-pair) were observed.

An embodiment of the invention is a composition and a method for usingTALENs to genetically modify livestock such as artiodactyls orzebrafish, dogs, mice, rats, fish, avian, chicken, or a laboratoryanimal. Many of the problems making these animals using conventionalprocesses have been discussed above. The genetic modification may be,for example, chosen from the list consisting of an insertion, adeletion, insertion of or change to an exogenous nucleic acid fragment,an inversion, a translocation, interspecies allele migration,intraspecies allele migration, and gene conversion to a natural,synthetic, or a novel allele. For instance, an undesired mutation in achromosome or chromosome pair may be replaced with a normal sequence. Ingeneral, a target DNA site is identified and a TALEN-pair is createdthat will specifically bind to the site. The TALEN is delivered to thecell or embryo, e.g., as a protein, mRNA or by a vector that encodes theTALEN. The TALEN cleaves the DNA to make a double-strand break that isthen repaired, often resulting in the creation of an indel, orincorporating sequences or polymorphisms contained in an accompanyingexogenous nucleic acid that is either inserted or serves as a templatefor repair of the break with a modified sequence. An exogenous sequencerefers to a sequence used to change the target cell, regardless ofwhether the sequence is actually a nucleic acid inserted intochromosomal DNA or if the sequence is used as a template to change thecellular DNA. The term nucleic acid fragment is broad and includes achromosome, expression cassette, gene, DNA, RNA, mRNA, or portionthereof. The term ssDNA includes ss-oligonucleotides. The cell or embryomay be, for instance, chosen from the group consisting of livestock, anartiodactyl, a cow, a swine, a sheep, a goat, a bird, a chicken, arabbit, and a fish. One embodiment is directed to a composition or amethod of making a genetically modified livestock and/or artiodactyl ora zebrafish, dogs, mice, bird, fish, avian, chicken, rats or alaboratory animal comprising introducing a TALEN-pair into livestockand/or an artiodactyl cell or an embryo that makes a geneticmodification to DNA of the cell or embryo at a site that is specificallybound by the TALEN-pair, and producing the livestockanimal/artiodactyl/other animal from the cell. Direct injection may beused for the cell or embryo, e.g., into a zygote, blastocyst, or embryo.Alternatively, the TALEN and/or other factors may be introduced into acell using any of many known techniques for introduction of proteins,RNA, mRNA, DNA, or vectors. Genetically modified animals may be madefrom the embryos or cells according to known processes, e.g.,implantation of the embryo into a gestational host, or various cloningmethods. The phrase “a genetic modification to DNA of the cell at a sitethat is specifically bound by the TALEN”, or “at a targeted chromosomalsite”, or the like, means that the genetic modification is made at thesite cut by the nuclease on the TALEN when the TALEN is specificallybound to its target site. The nuclease does not cut exactly where theTALEN-pair binds, but rather at a defined site between the two bindingsites.

Another such embodiment involves a composition or a treatment of a cellthat is used for cloning the animal. The cell may be of a livestockand/or artiodactyl cell, fish, zebrafish, dog, mice, rat, laboratoryanimal, bird, fish, chicken, a cultured cell, an immortalized cell, aprimary cell, a primary somatic cell, a zygote, a germ cell, aprimordial germ cell, a blastocyst, or a stem cell. For example, anembodiment is a composition or a method of creating a geneticmodification comprising exposing a plurality of primary cells in aculture to TALEN proteins or a nucleic acid encoding a TALEN or TALENs.The TALENs may be introduced as proteins or as nucleic acid fragments,e.g., encoded by mRNA or a DNA sequence in a vector.

Genetic modification of animals may also include transfection with areporter. As discussed above, primary cells were observed to be unstableas a result of cellular modifications mediated by the TALENs and/orTALENs introduction. As a result, success in the modification of primarycells (among other cell types), and/or the creation of new lines oflivestock from such cells is not reasonably expected using conventionalmeans. It is theorized, without being bound to a specific theory thatcells that express a gene cassette from a first vector are also likelyto be successfully modified by a TALEN delivered independently by mRNAor another vector. Expression of a reporter at the embryo/cell-levelmodification stage allows for elimination of cells that do not expressthe reporter. Alternatively, it allows for moving cells that express thereporter from the culture for use in animals by cloning or othertransgenic animal techniques, or into a second culture for furthercultivation and/or expansion in number and/or addition of furthervectors and/or nucleic acids and/or TALENs and/or other geneticmodifications. Selecting cells based on their expression of a reporterthat is independent of the gene of interest is a type of co-selectionprocess.

The term reporter, as used herein, refers to genes or transgenes thatencode reporters and selection markers. The term selection marker, asused herein, refers to a genetically expressed biomolecule that confersa trait that permits isolation by either positive or negative survivalselection criteria. The reporter may be, e.g., a fluorescent marker,e.g., green fluorescent protein and yellow fluorescent protein. Thereporter may be a selection marker, e.g., puromycin, ganciclovir,adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418,APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase,thymidine kinase (TK), or xanthin-guanine phosphoribosyltransferase(XGPRT). Phenotypic markers are markers based on discernible physicaltraits (e.g., epitopes or color), growth rate, and/or viability.

The term selection marker, as used herein, refers to a geneticallyexpressed biomolecule that confers a trait that permits isolation byeither positive or negative survival selection criteria. The reportermay be, e.g., a florescent marker, e.g., green fluorescent protein andyellow fluorescent protein. The reporter may be a selection marker,e.g., puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR),hygromycin-B-phosphtransferase, thymidine kinase (TK), orxanthin-guanine phosphoribosyltransferase (XGPRT). For instance, aselection marker may allow a cell to survive in the presence of a smallmolecule, thereby enabling selection. Other phenotypic markers may beused to select animals, such markers are based on discernible physicaltraits (e.g., epitopes or color), growth rate, and/or viability.

Embodiments of the invention include introducing a reporter (forinstance by use of a vector) and a TALEN (e.g., by an independent vectoror mRNA) into a cell or embryo. The cell may be from a livestock and/orartiodactyl, bovine, avian, chicken, zebrafish, dog, mice, rats or alaboratory animal. The TALEN and/or reporter may be directly introduced,e.g., by injection, or other means, e.g., involving cell culture. A cellculture may be made comprising cultured cells (primary cells, zygotes,oocytes, immortalized cells, germ cells, primordial germ cells, stemcells), a first nucleic acid encoding a TALEN, e.g., mRNA or a vectorwith DNA encoding the TALEN, and an independent vector having a DNAsequence encoding a reporter. The mRNA or first vector do not encode anyreporters and the second vector does not encode any TALs and does notencode any TALENs.

Vectors for the reporter, selection marker, and/or one or more TALEN maybe a plasmid, transposon, transposase, viral, or other vectors, e.g., asdetailed herein. Transposases may be used. One embodiment involving atransposases provides a vector that encodes a transposase. Other vectorsencode a transposon that is recognized by the transposase and has anucleic acid fragment of interest, e.g., a reporter, selection marker,exogenous nucleic acid for insertion or as a template for modification,or one or more TALENs. Accordingly, a cell or embryo may be transfectedwith a number of vectors between, for example, 1 and about 6; artisanswill immediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated, e.g., 2, 3, 4, 5, and 6. Morevectors may be used. The reporter may be used to identify cells that arelikely to have undergone modification by TALENs. Or a selection markermay be used to enrich the proportion of TALEN-modified cells bydestroying cells or embryos that do not express the selection marker.

An embodiment of the invention is a cell or embryo culture exposed to,or injected with, a plurality of vectors. A first vector comprises aTALEN or TALEN-pair; alternatively there are two TALEN vectors thatindependently provide a left TALEN and a right TALEN. A second vectorcomprises a reporter. The reporter may provide for non-destructiveidentification or may be a selection marker. A vector encoding aselection marker may be used as an alternative to the reporter vector,or in addition to the reporter vector. A further vector may encode anexogenous nucleic acid.

A process for making TALEN-modified cells, embryos, or animals cancomprise assaying a cell or embryo exposed to a TALEN for expression ofa reporter and using that cell or embryo in a method or composition formaking a genetically modified livestock and/or artiodactyl or otheranimal (fish, zebrafish, dogs, mice, avian, chicken, rats or alaboratory animal). For instance, a primary cell may be removed from acell culture and used for cloning. Or, a primary cell may be removedfrom culture and placed in a second culture to make a clonal line or forfurther processes. Or, an embryo or zygote expressing the reporter maybe used for either implantation into a surrogate dam or can be used forcloning, while other embryos or zygotes that do not express the reporternot used for cloning. In some embodiments, the reporter is a selectionmarker that is used to select for cells or embryos that express themarker.

Allele Introgression in Pig, Goat and Cattle Genomes

While plasmid templates were effective for introgression of POLLED andGDF8, the inventors believe that many desirable alleles correspond toSNPs. A set of experiments used oligonucleotide templates that had anoverlap in their cognate TALEN-binding sites and that also introduced a4 bp indel into the spacer region for restriction fragment lengthpolymorphism (RFLP) analysis. Primary fibroblasts were transfected withplasmid- or mRNA-encoded TALENs plus oligo templates and incubated 3days at either 30 or 37° C. TALENs delivered as mRNA consistentlyoutperformed plasmid in cells incubated at 30° C. (FIG. 37A). Despiteappreciable levels of TALEN activity measured by the SURVEYOR assay, HDRwas consistently higher (>2-fold) when TALENs were delivered as mRNAcompared to plasmids. This observation was surprising, and it wasspeculated that could have been a result of favorable kinetics; e.g.,TALENs from mRNA were more rapidly translated allowing utilization ofthe template prior to oligo degradation. However, a time-courseexperiment showed little difference in the onset of HDR between TALENsencoded by plasmid versus mRNA (FIG. 37B). Among replicates using TALENmRNA at 30° C., the levels of cumulative mutation and total HDR weresimilar, suggesting the majority of mutant alleles corresponded to theintended introgression.

In some studies, TALEN-induced indels declined 50-90% after extendedculture where selection processes or markers were not used (Carlson, D.F. et al. Efficient TALEN-mediated gene knockout in livestock,Proceedings of the National Academy of Sciences, 109:17382-17387 (2012),herein “Carlson 2012”). In other words, in some instances, when indelswere made, they were often not stable and a selection marker process wasused to identify stable changes. In contrast, it was observed hereinthat HDR levels at four loci were roughly equivalent when measured atdays 3 and 10 without selective enrichment, indicating that these HDRindel alleles were stable in culture (FIG. 37A). The consistently highrate (25-50%) and stability of gene edits at all four loci suggestedthat edited cells should be recoverable by dilution cloning withoutselective enrichment, reporters or selection markers. Furtherexperimental work involving analysis of about 1,000 colonies for definedindel alleles in eight separate loci revealed a recovery rate of 10-65%(average 42%) where up to 32% of the colonies are homozygous for theintended edit (Table 7). The data shows that introducing TALENs as mRNAinto the cell is helpful for efficiency and stability; extended culturetimes at 30° C. were also helpful.

Production of Biomedical Model Animals with Gene-Edited Alleles

Two gene-edited loci in the porcine genome were selected to carrythrough to live animals—APC and DAZL. Mutations in the adenomatouspolyposis coli (APC) gene are not only responsible for familialadenomatous polyposis (FAP), but also play a rate-limiting role in amajority of sporadic colorectal cancers. Dazl (deleted inazoospermia-like) is an RNA binding protein that is important for germcell differentiation in vertebrates. The DAZL gene is connected tofertility, and is useful for infertility models as well asspermatogenesis arrest. Colonies with HDR-edited alleles of DAZL or APCfor were pooled for cloning by chromatin transfer. Each pool yielded twopregnancies from three transfers, of which one pregnancy each wascarried to term. A total of eight piglets were born from DAZL modifiedcells, each of which reflected genotypes of the chosen coloniesconsistent with either the HDR allele or deletions resulting from NHEJ.Three of the DAZL piglets were stillborn. Of the six piglets from APCmodified cells, one was stillborn, three died within one week, andanother died after 3 weeks, all for unknown reasons likely related tocloning. All six APC piglets were heterozygous for the intendedHDR-edited allele and all but one either had an in-frame insertion ordeletion of 3 bp on the second allele. Remaining animals are beingraised for phenotypic analyses of spermatogenesis arrest (DAZL−/−founders) or development of colon cancer (APC+/− founders).

Template-driven introgression methods are detailed herein. Embodimentsof the invention include template-driven introgression, e.g., by HDRtemplates, to place an APC or a DAZL allele into a non-human animal, ora cell of any species.

This method, and methods generally herein, refer to cells and animals.These may be chosen from the group consisting of vertebrate, livestock,an artiodactyl, a primate, cattle, a swine, a sheep, a goat, a bird, achicken, a rabbit, fish, dog, mice, rat, cat or laboratory animal. Theterm livestock means domesticated animals that are raised as commoditiesfor food or biological material. The term artiodactyl means a hoofedmammal of the order Artiodactyla, which includes cattle, deer, camels,hippopotamuses, sheep, pigs and goats that have an even number of toes,usually two or sometimes four, on each foot.

Alleles for Introgression

Allele introgression has many important applications. The AllelicIntrogression Table, below, and Table 7 (Frequencies for recovery ofcolonies with HDR alelles) describe certain genes and theirapplications. Artisans reading this application will be able to make anduse the introgressions and resultant cells and animals. Artisans canreadily apply the processes set forth herein for the use of thesealleles as templates or targets for disruption. Embodiments includemaking a genetically modified cell or animal (for instance, a labanimal, an F0 founder, or animal line) whose genome has received a genefrom Table 7 or the Allelic introgression Table, e.g., by insertion ortemplate-driven allele introgression that replaces the endogenous allelewith an allele from Table 7 or the Allelic introgression Table. Allelesfor some genes are reported to provide livestock production advantages,but are at very low frequencies or are absent in some breeds or species(see items 1-9). Introgression of these alleles can be of significantvalue for production traits. For example, the Polled allele (item 1)from beef breeds results in animals that do not have horns, whereasdairy breeds do not have this allele so have horns and need to bedehorned as a production practice. Allele introgression from beef breedsinto horned (dairy) breeds will lead to hornless dairy cattle which ishas value for both production and animal welfare. Other examples relateto alleles that can increase or enhance characteristics of agriculturalproducts such as meat (items 4-6) and milk (items 7-8). Item 9 is usefulfor disease resistance.

Many commercial and commonly used animal breeds have been carefully bredto establish desirable traits but, in the process of that breeding, haveaccumulated genetic errors that reduce their reproductive successbecause of losses in fertility or by increasing miscarriages.Deleterious alleles for some genes are present in animal populations. Asexplained elsewhere herein, the inventive techniques provide forchanging alleles only at an intended location in a target animal,without other modifications resulting from genetic tools or from meioticrecombinations. Therefore, for the first time, it is possible toclean-up the genetic errors that have accumulated in livestock andanimal breeds without disrupting the genome of the animals and,consequently, disrupting traits or causing unintended consequences.Alleles for some genes can be used to control animal fertility forgenetic control of breeding stock (items 2-3). The term breed is a termof art that refers to domestic animals that, through selection andbreeding, have come to resemble one another and pass those traitsuniformly to their offspring. The animals that belong to a particularbreed are known to artisans that practice in these arts. Breed specificcharacteristics, also known as breed traits, are inherited, and purebredanimals pass such traits from generation to generation. Thus, allspecimens of the same breed carry several genetic characteristics of theoriginal foundation animal(s). In order to maintain the breed, a breederwould select those animals with the most desirable traits to achievefurther maintenance and developing of such traits. At the same time, thebreed would avoid animals carrying characteristics undesirable or nottypical for the breed, including faults or genetic defects. In ourexamples, we recruit genetic benefits (specific traits) from one breedinto another without the traits that are considered negative in acertain breed.

Many useful animal models can be made. Certain alleles are useful, seeAllelic introgression Table items 10-39. Some of these are establishedin animals. Others of the genes are known to cause human disease, sointrogressing these alleles into livestock, lab animals, or otheranimals is useful to create biomedical models of human disease.

Embodiments of the invention include a method of making a geneticallymodified animal, said method comprising exposing embryos or cells to anmRNA encoding a TALEN, with the TALEN specifically binding to a targetchromosomal site in the embryos or cells, cloning the cells in asurrogate mother or implanting the embryos in a surrogate mother, withthe surrogate mother thereby gestating an animal that is geneticallymodified without a reporter gene and only at the TALEN targetedchromosomal site wherein the allele is a member of the group consistingof (a) horn polled locus (b) a gene recessive for fertility defects,e.g., CWC15 and/or ApaF1 (c) genes for enhancing a livestock trait,e.g., meat production (GDF8, IGF2, SOCS2, or a combination thereof)and/or milk production (DGAT1 and/or ABCG2) (d) a gene for changinganimal size (PLAG1, GHRHR) (e) genes that potential tumor growth (e.g.,TP53, APC, PTEN, RB1, Smad4, BUB1B, BRCA1, BRCA2, ST14 or a combinationthereof) (f) human oncogenes for animal models of cancer (e.g., AKT1,EGF, EGFR, KRAS, PDGFRA/B or a combination thereof) (g) genes in animalmodels for hypercholesterolemia (to induce atherosclerosis, stroke, andAlzheimer's disease models), e.g., LDLR, ApoE, ApoB or a combinationthereof (h) Inflammatory Bowel disease, e.g., NOD2 (i) spina bifida,e.g., VANGL1 and/or VANGL2 (j) pulmonary hypertension, e.g., miR-145 (k)genes for cardiac defects, e.g., BMP10, SOS1, PTPN11, Nrg1, Kir6.2,GATA4, Hand2, or a combination thereof and (1) celiac disease genes,e.g., HLA-DQA1.

Allelic Introgression Table Genes; Species Item [Gene ReferenceIdentification] Application 1 Horn-Polled Locus; Bovine Transfer alleleinto cows of various breeds to make [UMD3.1:1:1705490:1706389:1] bovinelines of those species without horns; see Medugorac, I., D. Seichter, etal., (2012). “Bovine polledness - an autosomal dominant trait withallelic heterogeneity.” PloS one, 7(6):e39477. 2 CWC15 (JH1) Use naturalallele as template to restore wildtype [hs Gene ID: 51503] sequence toanimal lines and breeds with defective 3 ApaF1 (HH1) alleles; seeVanRaden, P. M., K. M. Olson, et al., [hs Gene ID: 317] (2011). “Harmfulrecessive effects on fertility detected by absence of homozygoushaplotypes.” J Dairy Sci., 94(12):6153-6161. 4 GDF8 Enhancement ofgrowth for meat production. [hs Gene ID: 2660] 5 IGF2 [hs Gene ID: 3481]6 SOCS2 [hs Gene ID: 8835] 7 DGAT1 Alleles of these genes are known toinfluence the [hs Gene ID: 8694] amount and composition of milk. 8 ABCG2Hs Gene ID: 9429] 9 GHRHR Size reduction of animals for Biomedicalmodeling. [hs Gene ID: 2692] 10 TP53 Tumor suppressor genes;heterozygous knockout to [hs Gene ID: 7157] potentiate tumor growth. 11APC [hs Gene ID: 324] 12 PTEN [hs Gene ID: 5728] 13 RB1 [hs Gene ID:5925] 14 Smad4 [hs Gene Id: 4089] 15 BUB1B [hs Gene ID: 701] 16 BRCA1[hs Gene ID: 672] 17 BRCA2 [hs Gene ID: 675] 18 ST14 [hs Gene ID: 6768]19 AKT1 Oncogenes. Activated human alleles will be [hs Gene ID: 207]introgressed into pigs to model cancers. 20 EGF [hs Gene ID: 1950] 21EGFR [hs Gene ID: 1956] 22 KRAS [hs Gene ID: 3845] 23 PDGFRA/B [hs GeneIDs: 5156/5159] 24 LDLR Hypercholesterolemia to induce atherosclerosis,[hs Gene ID: 3949] stroke and Alzheimer's disease models. 25 ApoE [hsGene ID: 348] 26 ApoB [hs Gene ID: 338] 27 NOD2 Inflammatory Boweldisease for animal models. [hs Gene ID: 641271] 28 VANGL1 Spina Bifidais associated with alleles of these genes. [hs Gene ID: 81839] Transferof these alleles in livestock will generate 29 VANGL2 models forbiomedical research. [hs Gene ID: 57216] 30 miR-145 Pulmonaryhypertension is associated with alleles [hs Gene ID: 611795] of thesegenes. Transfer of these alleles in swine will generate models forbiomedical research. 31 BMP10 Cardiac defects associated with alleles ofthese [hs Gene ID: 27302] genes. Transfer of these alleles will generate32 SOS1 models for biomedical research. [hs Gene ID: 6654] 33 PTPN11 [hsGene ID: 5781] 34 Nrg1 [hs Gene ID: 3084] 35 Kir6.2 [hs Gene ID: 3767]36 GATA4 [hs Gene ID: 2626] 37 Hand2 [hs Gene ID: 9464] 38 HLA-DQA1Alleles associated with celiac disease will be [hs Gene ID: 3117]transferred to livestock to create an animal model.Differential Stability of Gene-Edits

It was not known if it was possible to have introgression of stable SNPsby NHEJ or HDR. As indicated in Table 7, both day-3 levels of HDR(7-18%) and the isolation of cellular clones with the intended SNPalleles (3-15%) within cattle and swine GDF8 or pig p65 wassignificantly lower than for indel alleles, where HDR ranged from 10 toabout 50%. This data suggested a hypothesis that indels were more stablethan SNP because the introduction or elimination of at least 4 bp in theTALEN spacer region would be expected to reduce re-cleavage of thelocus, consistent with constraints on TALEN spacer length. And even a 4bp insertion allele was more efficient than SNP alleles, basedcomparison of HDR frequencies with oligo within the same locus suggested(FIG. 38). This hypothesis also explained why sequence analysis revealedthat nearly half of the isolated SNP-positive colonies for GDF8 or pigp65 harbored concomitant indels expected to change TALEN spacing.Regardless, it was possible to recover colonies with homozygousconversion of G938A in GDF8 (both pigs and cattle) and T1591C in pig p65at up to nearly a 5 percent level without any additional changes to thelocus (Table 7). It was also possible to introgress small polymorphismsfor the sheep FecB and Callipyge loci into the goat genome. This abilityto precisely alter a single nucleotide or 1-3 nucleotides is surprisingas well as significant. As a comparison, it was also possible to designCRISPR gRNAs that overlapped the T1591C site of p65 and to compareintrogression using the two platforms. Despite efficient production ofDSB at the intended site, CRISPR/Cas9-mediated HDR was lower than 6percent at day-3 and below detection at day-10 (FIG. 39). Recovery ofmodified clones with CRISPR-mediated HDR was also lower than with TALENseven though the TALENcleavage site was 35 bp away from the SNP site(Table 7). Analysis of CRISPR/Cas9 induced targeting at a second locus,ssAPC14.2, was much more efficient, but still did not reach the level ofHDR induced by TALENs at this site, circa 30 versus 60% (FIG. 40).

Strategies for Introgression of Alleles and for Stabilizing IntrogressedSNP Alleles

Given the conservation of the 5′-thymidine nucleotide immediatelypreceding TAL-binding sites, it was reasoned that altering these basesin the oligo HDR template (referred to as blocking mutations (BM)) wouldinhibit re-cleavage of edited alleles. Surprisingly, the BMs had nosignificant impact on the maintenance of SNP alleles at the pig LDLR orGDF8 loci (FIG. 41 panel a). This suggested that either the conversiontract for oligo-templated HDR is quite short and does not incorporatethe BM, or that altering the 5′-thymidine does not completely abolishTALEN activity.

ILLUMINA deep sequencing was conducted for 200-250 bp amplicons flankingthe target sites from populations of cells transfected with oligos andTALEN mRNA. The results from five loci in pigs and cattle showed thatinsertion alleles were in general more prevalent and stable in thepopulation (FIG. 42). Whereas BMs had little influence on thepreservation of intended alleles in culture, there was a slight biastowards incorporation of BMs in SNP edited alleles versus insertionaledits (FIG. 43). with our colony analysis, reads sorted on the basis ofincorporating the intended SNP (iSNP), G938A or T1591C conversion inbtGDF8 and p65, revealed that nearly half of reads with the iSNP had anadditional mutation (iSNP+Mut) (FIG. 42 panel b), the majority of whichwere indels (FIG. 43). The majority of iSNP btGDF8 reads with indels inthe spacer also contained one or both BM (data not shown) demonstratingthat modification of the conserved 5′-thymidine was not able to suppressre-cleavage and subsequent indel generation. Thus, this base must beless critical to TALEN-binding than suggested by conservation, andprovides a molecular basis for the inability of BMs to preserve allelesas described above.

Another strategy to reduce re-cutting of the SNP edits is to designTALENs such that their binding sites overlap the target SNPs. Theinfluence of RVD/nucleotide mismatches within the TALEN-binding site forintrogression of G938A SNP into cattle GDF8 was evaluated. Two pairs ofTALENs were generated, one that bound the wildtype “G” allele(btGDF83.6-G) and another that bound the intended “A” allele(btGDF83.6-A) (FIG. 41 panel b). HDR with each TALEN pair was similar atday-3 whereas levels measured at day-12 were significantly higher usingthe TALENs that bound the wildtype “G” allele, indicating thatrecleavage was more prevalent with btGDF83.6-A which targets therepaired allele perfectly. Different RVD/nucleotide mismatches may havegreater influence on maintenance of HDR alleles since the NN-RVD usedfor the wildtype “G” TALENs is able to bind both G and A nucleotides.For modification of porcine EIF4GI, it was found that threeRVD/nucleotide mismatches were sufficient for protection of the HDR-editas nearly 70% of isolated colonies contained an edited allele, greaterthan half of those being homozygotes (Table 7 and FIG. 44). Thus, theintentional alteration of the target locus to resist recleavage is aneffective strategy for preserving edits.

It was hypothesized that gene-editing is a dynamic process. TALENcleavage and re-cleavage are theorized to be in flux with repair byNHEJ, HDR with oligo template, and HDR with the sister chromatid astemplate. It was hypothesized that the observed loss of SNP allelesmight be reduced by extending the hypothermic treatment, slowing cellproliferation long enough to outlast the burst of TALEN activity fromTALEN mRNA transfection. Indeed, and surprisingly, this extension almosttripled the level of SNP HDR-edited alleles recovered after extendedculture (FIG. 45).

For biomedical applications, alterations of bases besides the key basesthat create the desired functionality is acceptable so long as thedesired phenotype is achieved and the other changes are apparentlywithout functional relevance. The inventors believe, however, that it isdesirable for animals used in agriculture, to duplicate natural (native)alleles without further changes or to make only the intended editswithout further changes. In contrast to the approaches where themismatches are derived from successful introgression of the HDRconstruct, mismatches can be derived from changes in the RVD sequence.For TALENs, this process requires the TALEN monomers to be constructedwith RVDs that do not bind to their corresponding nucleotides in thenative alleles (FIG. 46 panel c). This concept of an intentionalmismatch in the design of the nuclease (in this case TALENs) to preventre-cutting of a desired is novel and operates under the followingtheory. TALENs can only tolerate some mismatches in their bindingregions before their binding activity is essentially eliminated. Thus,it is possible to develop TALENs that have intentional mismatches withthe native allele that will still cut, but as more mismatches arecreated, binding will be abolished. The theory of intentional mismatchis that after introgression of the desired allele, the new mismatch willhave an additive effect to the engineered mismatches in the TALEN codeto pass a critical tipping point to render the TALEN inactive.Counterintuitively, decreasing nuclease affinity for a target byintentional mismatching of RVDs provides a strategy to introgress aspecific mutation down to a single nucleotide polymorphism (SNP), andreduce to undesired indels as a result of re-cutting.

As an example, a TALEN pair (caCLPG 1.1) was designed with zeromismatches to target the CLPG locus in the goat (Capra aegagrus hircus)genome (FIG. 46 panel a). The desired mutation was an adenine to guanineSNP, which has been linked to an increase in hindquarter musclehypertrophy. The SNP allowed easy genotyping of colonies due to a lossof an AvaII restriction site. Initial restriction digest assays showedseveral colonies with promising results, however when the alleles ofeach colony were sequenced, zero of fourteen were our intended productas each contained an undesired indel in addition to the desired SNP(FIG. 46 panel b and Table 6 labeled Success rate using intentionalmismatches). To test the intentional mismatch approach, an additionalthree TALEN pairs were developed with different numbers and locations ofintentional mismatches (FIG. 46 panel c). These TALENs were able to cutthe wild-type allele at similar frequencies to the original caCLPG1.1TALEN pair (FIG. 46 panel d). To observe the effect on HDR and reducingof undesired indels, individual colonies were derived from cellstransfected with caCLPG 1.1c (two mismatches) and the HDR template. Incontrast to the results with the original caCLPG1.1 TALENs, three offifteen (20%) of AvaII resistant colonies were positive for the desiredSNP and contained no additional mutations (3/15=20%) (FIG. 46 panel eand Table 6). Thus, derivation of the intended allele was dependent onintentional mismatch. TALEN pair btPRLR 9.1 (see Tables 6, 10, and 14)was used to cause a frameshift in the bovine prolactin receptor gene(see FIGS. 66 and 67).

TABLE 6 Success Rate Using Intentional Mismatch TABLE 6: Success ofintentional Mismatch and HDR at Agriculturally relevant loci. Number ofSpecies/Cell Allele RVD Gene type desired mismatches TALEN ID RFLP pos.Confirmed CLPG Goat/ A to G 0 caCLPG1.1 NA 0/14 Fibroblasts CLPG Goat/ Ato G 2 caCLPG1.1c NA 3/15 Fibroblasts DGAT Cow/ K232A 0 btDGAT 14.219/96 0/12 Fibroblasts DGAT Cow/ K232A 1 btDGAT 14.4 15/96 0/12Fibroblasts DGAT Cow/ K232A 1 btDGAT 14.5 16/96 2/12 Fibroblasts DGATCow/ K232A 1 btDGAT 14.6 15/96 3/12 Fibroblasts PRLR Cow/ Trunc461 0btPRLR 9.1 NA 2/11 Fibroblasts SOCS2 Pig/ Trunc10 0 ssSocs 2.1 75%^(b)Fibroblasts SLC35A3 Cow/ V180F^(a) 0 SLC35A3 8.3 18%^(b) Fibroblasts^(a)Repair of the missense allele that results in complex vertebralmalformation (Thomsen, B; Genome Res. 2006 Jan; 16(1): 97-105.)^(b)Percentage of HDR on the population level CLPG (callipyge gene);DGAT (Diacylglycerol O-Acyltransferase); PRLR (Prolactin Receptor);SOCS2 (Suppressor of cytokine signaling 2); SLC35A3 (Solute CarrierFamily 35 Member A3)

In a further example, the intention was to alter specifically two basesin the bovine DGAT gene. As with the CLPG locus, attempts to introgressthe desired mutation without intentional mismatch failed as 0/12 RFLPcolonies that were positive for the HindIII site were free of indels(FIG. 47 panel d and Table 6). The intentional mismatch method was used,and found overall rates of HDR on the population level (FIG. 47 panelc). Sequencing from individual colonies however revealed that two ofthree of the intentional mismatch designs were successful, giving riseto 2/12 and 3/12 correct alleles for 14.5 and 14.6 respectively (FIG. 47panel e and Table 6). As with the CLPG locus, derivation of the intendedallele was dependent on RVD-encoded intentional mismatch. The data inTable 7 demonstrated that combining mRNAs encoding TALENS plusoligonucleotides as templates for directing HDR achieved severalbenchmarks for a genome-editing strategy: 1) only target nucleotideswere changed and mRNA transfection avoided unintended integration ofplasmid DNA, 2) gene edits were efficient; from about 10% for SNPs toabove 50% for some larger alterations, and 3) the method was reliable;targeted alteration of 16/16 sites (15 genes) was achieved. Theefficiency and precision reported here is very surprising.

TABLE 7 Frequencies for recovery of colonies with HDR alleles Mutationaa Day 3% Bi-allelic Reagent ID Species type at change change HDR HDR+(%) HDR+ (%) TALEN ssLDLR2.1³ Pig ♀ Ins/FS 141(Ins4) 47ΔPTC 38 55/184(30)  4/184 (2)  TALEN ssDAZL3.1⁴ Pig ♂ Ins/FS 173(Ins4) 57ΔPTC 25 34/92(37) 8/92 (9) TALEN ssDAZL3.1^(Rep) Pig ♂ Ins/FS 173(Ins4) 57ΔPTC 3042/124 (34)  7/124 (6)  TALEN ssAPC14.2^(b) Pig ♂ Ins/FS 2703(Ins4)902ΔPTC 48 22/40 (55)  4/40 (10) TALEN ssAPC14.2^(Rep) Pig ♂ Ins/FS2703(Ins4) 902ΔPTC 50 57/96 (60) 19/96 (20) TALEN ssAPC14.2^(Ld) Pig ♂Ins/FS 2703(Ins4) 902ΔPTC 34 21/81 (26) 1/81 (1) TALEN ssTp53 Pig ♂Ins/FS 463(Ins4) 154ΔPTC 22 42/71 (59) 12/71 (17) TALEN ssRAG2.1 Pig ♂Ins/FS 228(Ins4) 76ΔPTC 47 32/77 (42) 13/77 (17) TALEN btRosa1.2^(c) Cow♂ Ins/mloxP Ins34 NA 45 14/22 (64)  7/22 (32) TALEN ssSRY3.2 Pig ♂Ins/mloxP Ins34 NA 30 ND ND TALEN ssKissR3.2 Pig ♂ Ins/FS 322(Ins6)107ΔPTC 53 57/96 (59) 17/96 (18) 323(del2) TALEN btGDFB3.1 Cow ♂ del/FS821(del11) FS ~10  7/72 (10) 2/73 (3) TALEN ssEIF4GI14.1 Pig ♂ SNPsG2014A N572D 52 68/102 (67)  40/102 (39)  T2017C C2019T L673F TALENbtGDFB3.6N Cow ♂ SNPs G938A C313Y 18 8/94 (9) 3/94 (3) T945C TALENbtGDFB3.6N^(d) Cow ♂ SNP G938A C313Y NA 7/105 (7)  2/105 (2)  TALENssP65.8 Pig ♂ SNP T1591C S531P 18  6/40 (15) 3/40 (8) TALENssP65.8^(Rep) Pig ♂ SNP T1991C S531P 7  9/63 (14) 3/63 (8) TALENssGDF83.6^(d) Pig ♂ SNP G938A C313Y NA 3/90 (3) 1/90 (1) TALEN caFecB6.1Goat ♂ SNP A747G Q249R 17 17/72 (24) 3/72 (4) TALEN caCLPG1.1 Goat ♂ SNPA→G Non- 4 ND ND coding CRISPR ssP68 G1s Pig ♂ SNP T1591C S531P 6 6/96(6) 2/96 (2) CRISPR ssP65 G2a Pig ♂ SNP T1591C S531P 3 2/45 (4) 0/45CRISPR APC14.2 G1a Pig ♂ Ins/FS 2703 (Ins4) 902ΔPTC 32 ND ND

Two concerns in gene editing are stabilizing the changes at the targetedsite and avoiding modification of unintended sites. With regard to thefirst, evidence was found that HDR-edits directing single basepairchanges, i.e., SNPs, could be lost (FIG. 41 and FIG. 42 panel b). Basedon the prediction that a thymidine preceding the targeted DNA sequenceinfluences TAL binding, it was attempted to block re-cleavage ofintrogressed alleles by introducing BMs. However, it was found that BMsdid not prevent TALEN activity and re-cleavage of edited alleles (FIG.41 and FIGS. 43 and 44). In contrast, introduction of multiple SNPs oradditional sequence (FIG. 37A and FIG. 44) resulted in more stableHDR226 edits. Surprisingly, it was found that extension of hypothermicculture resulted in the stabilization of introgressed SNP alleles. It istheorized that hypothermia slows cell proliferation primarily byprolonging G1-phase of the cell cycle so that this treatmentdifferentially favors oligo-HDR versus sister chromatid templated repairin a cell-cycle dependent manner. Regardless of the mechanism, thisapproach offers a straight-forward strategy for recovering cells withprecise introgression of SNP alleles.

A variety of objectives were achieved by precise gene editing (Table 7).Knockout of genes of biomedical relevance was accomplished byinterrupting coding sequences with 4 bp indels. This strategy wasreliable and generally resulted in HDR-edits in about 40% of the clones(range 26-60%), and of those, up to one-third were homozygotes. Atsimilar frequencies, a modified loxP (mloxP) site was integrated intoROSA26 and SRY loci in cattle and pigs that can be used as a landing pad(also referred to as a safe harbour) for insertion of novel sequences inlivestock via recombinase-mediated cassette exchange. Previously, onlyNHEJ edits had been demonstrated for the Y chromosome of livestock,however, TALENs are suitable for direct stimulation ofknockout/knock-in, at least as demonstrated in mice. Also, there was anintrogression of native alleles between species/breeds, including thedouble-muscling mutations of GDF8 (SNP G938A23, 25 or 821del1123-25 fromPiedmontese and Belgian Blue cattle respectively) into the genome ofWagyu cattle and Landrace pigs.

In some cases gene targeting with a standard plasmid vector andhomologous recombination cassette will not be suitable for transgenedelivery. Some cases could include when attempting to place a transgenein a site surrounded by repetitive elements or low complexity DNA. Inthese cases, the short homology required by oligo HDR may be preferredto integrate a transgene into a small region of unique sequence.However, the cargo capacity for oligo HDR is not sufficient to deliver atransgene. To circumvent this problem, we sought to combine theefficiency of oligo HDR for delivery of small insertions (e.g., LoxPsites) and the large cargo capacity of recombinase mediated cassetteexchange (RMCE) for site specific integration of transgenes.Recombinase-mediated cassette exchange (RMCE) is a method based on thefeatures of site-specific recombination processes (SSRs). This processallows for systematic, repeated modification of higher eukaryoticgenomes by targeted integration. This result is achieved with RMCE bythe clean exchange of a preexisting gene cassette for an analogouscassette carrying the gene of interest (GOI).

There are problems with using RMCE to make genetically modified animalsin the higher vertebrates, such as livestock. A significant problem isthat due to the short lifespan of primary livestock cells prior tosenescence, this process must occur in a single treatment. It would bepossible in some other types of cells to perform the RMCE processserially wherein a cellular clone with the inserted LoxP site isisolated prior to transfection with the RCME machinery and isolation ofclones to identify those with the correct targeting event. Applicantsattempted to perform this process by simultaneously transfecting primaryfibroblasts with four components: 1) SRY TALENs 2) an oligo withhomology to SRY that includes two RMCE compatible loxP sites 3) a RMCEcompatible transgene and 4) a source of Cre recombinase. In FIG. 48, theRMCE transgene was the puromycin resistance gene enabling selection forintegration events. The number of puromycin resistant colonies wassignificantly increased when YFC-Cre was provided in contrast to thecontrol group that included a mCherry expression cassette in place ofYFC-Cre. Among puromycin resistant colonies (selected from cells treatedfor 3 days at either 30° C. or 37° C.) eight (n=95) and four percent(n=95) were positive for correct targeting of the RMCE vector. Theseresults showed that it was possible to simultaneously provide theTALENS, HDR template containing loxP site, a transgene of interestflanked by loxP, and a Cre-recombinase mRNA resulting in RMCE mediatedrecombination into a TALEN targeted locus.

Embodiments of the invention include a process of homology dependentrepair using an HDR template with a sequence that is introduced into thehost cell or embryo that is a landing pad, e.g., for exogenous genes.The term landing pad is used according to its customary meaning to referto a site-specific recognition sequence or a site-specific recombinationsite that is stably integrated into the genome of a host cell. Presencein the host genome of the heterologous site-specific recombinationsequence allows a recombinase to mediate site-specific insertion of aheterologous polynucleotide or an exogenous into the host genome.

Embodiments include, kits, uses, compositions, and a method of creatinga landing pad in a chromosomal DNA of a cell, comprising introducing atargeted nuclease system and a HDR template into the cell, with thetargeted nuclease system comprising a DNA-binding member forspecifically binding an endogenous cognate sequence in the chromosomalDNA, wherein the targeted nuclease system and the HDR template operateto alter the chromosomal DNA to have identity to the HDR templatesequence, wherein the HDR template sequence comprises a landing pad. Themethod may be applied in a primary cell or embryo. Embodiments includeintroducing the targeting nuclease, the HDR template encoding thelanding pad, the exogenous gene that is compatible with the landing pad,and a source of recombinase compatible with the same; all of these maybe introduced simultaneously. The term simultaneous is in contrast to ahypothetical process of treating cells multiple times in seriatim; theterm must be kept in context, with an appreciation that it refers to aliterally simultaneous introduction or an introduction calculated tohaving all of the factors bioactive at the same time. The landing sitemay be, e.g., RMCE compatible loxP sites, FRT, rox, VloxP, SloxP. Therecombinase may be, e.g., Cre, FLP, Dre.

In other experiments, for improvement of animal welfare, the POLLEDallele was transferred from a beef producing breed into cells fromhorned dairy cattle. A candidate SNP allele for African swine fevervirus resilience (T1591C of p6539) was transferred from warthog to thegenome of conventional swine cells and introgressed sheep SNPsresponsible for elevated fecundity (FecB; BMPR-IB) and parent-of-origindependent muscle hypertrophy (Callipyge) were transferred into the goatgenome. Such introgression was previously impossible by breeding andwill enable the assessment of defined genetic effects in relatedspecies. Non-meiotic allele introgression has not conventionally beenpossible without selective enrichment, and efficiencies reported hereinare 10³-10⁴-fold higher than results previously obtained with selection.Such high levels of unselected single-allele introgression suggests itwill be feasible to alter multiple alleles in a single generation offarm animals, decreasing the impact of long generation intervals ongenetic improvement. Furthermore, efficient editing to homozygosity willgreatly increase the rate of introgression per breeding interval.

As further elaboration of inventions described here customizedendonucleases were used to generate live animals with precise edits attwo independent loci. Pigs edited to disrupt the DAZL gene are useful asa model for studying the restoration of human fertility by germ celltransplantation, or for the production of genetically modified offspringby transfer of genetically modified germline stem cells as demonstratedin pigs, goats, and rodents. Gene edited alleles of APC provide asize-relevant model of colon cancer for pre-clinical evaluation oftherapeutics, surgical intervention or detection modalities. Theseresults demonstrate an introduction of genetic modifications, includingpolymorphisms, and including modifications that mimic naturalpolymorphisms into livestock. Gene-editing technology is useful toaccelerate genetic improvement of agricultural products by intra- andinterspecific allele introgression to help meet the growing globaldemand for animal protein. It also is useful for the development oflarge animals with defined genetics for drug and device testing, or forthe development of therapeutic cells and organs. Other uses includemaking cells that can be used in vitro for research to understand themechanisms of congenital and infectious disease, and to improve themethods for gene editing and control.

Gross Chromosomal Deletions and Inversions; Genetically Modified Animals

Experiments were performed with TALENs directed to a plurality of DNAsites. The sites were separated by several thousand base pairs. It wasobserved that the DNA could be rejoined with the deletion of the entireregion between the sites. Embodiments include, for example, sitesseparated by a distance between 1-5 megabases or between 50% and 80% ofa chromosome, or between about 100 and about 1,000,000 basepairs;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., from about1,000 to about 10,000 basepairs or from about 500 to about 500,000basepairs. Alternatively, exogenous DNA may be added to the cell orembryo for insertion of the exogenous DNA, or template-driven repair ofthe DNA between the sites. Modification at a plurality of sites may beused to make genetically modified cells, embryos, artiodactyls, andlivestock. Example 5 describes the deletion of several thousandbasepairs of DNA, with rejoining of the ends verified biochemically.

Unexpectedly, TALEN-cleavage at separated sites also resulted infrequent inversion of the entire region between TALEN targets. As anadditional surprise, as detailed in Example 5, these inversions wereaccomplished with great fidelity. Forty one out of 43 of the testedinversions were positive at both the 5′ and 3′ junctions. And sequencingof PCR products confirmed both deletion and inversion events withaddition or deletion of very few nucleotides at their junctions (FIG.11, 12). This result was highly surprising and unprecedented. Cells orembryos with these deletions or inversions have many uses as assay toolsfor genetics.

These cells are also useful for making animals, livestock, and animalmodels. The term animal model includes, for example, zebrafish, dogs,mice, rats or other laboratory animals. Large deletions provide for geneinactivation. Also, for instance, a deletion strain may be made ofcells, livestock, or animal models. Crossing the deletion strains withan organism bearing a mutation for comparison to a wild-type helps torapidly and conveniently localize and identify the mutation locus.Deletion strains are well known in these arts and involve sets oforganisms made with a series of deletions in a gene. Deletion mappinginvolves crossing a strain which has a point mutation in a gene with thedeletion strains. Wherever recombination occurs between the two strainsto produce a wild-type (+) gene, the mutation cannot lie within theregion of the deletion. If recombination cannot produce any wild-typegenes, then it is reasonable to conclude that the point mutation anddeletion are found within the same stretch of DNA. This can be used forexample to identify causative mutations, or to identify polymorphismsunderlying quantitative trait loci.

Cells, embryos, livestock, artiodactyls, and animal models withinversions are also useful for fixing a genetic trait in progeny of anorganism or an animal line or animal breed. Recombinations typicallyoccur between homologous regions of matching chromosomes during meiosis.Inversion of a chromosomal region destroys homology and suppressesmeiotic recombination. Methods and compositions described herein may beused to make such organisms or animals. For example, DNA in a somaticbovine or porcine cell may be cut at a plurality of loci by TALENs, andcells with an inversion may be isolated, or cells expressing reportersmay be used as likely candidates for successful inversions. The cellsmay be used to clone animals that harbor chromosomal regions that areincapable of meiotic recombination. Alternatively, it is expected thatinversions will also occur at reasonable frequencies in embryos treatedwith multiple TALEN-pairs at plurality of sites.

An embodiment of this method is identifying a DNA region encoding agenetic trait and cutting a DNA in a cell or embryo on each side of theencoded trait at sites using a plurality of TALENs. The modified cell orembryo may be used for creating a genetically modified animal. Themethod may comprise isolating a genetically modified animal that has theinversion.

Animals Genetically Modified without any Reporters; TALENs Techniques;Allelic Migration

Certain embodiments of the invention are directed to processes ofmodifying cells or embryos without the use of reporters and/or selectionmarkers. In general, it was initially observed that the frequency ofTALEN-modified cells decreases significantly over time in the absence ofenrichment or selection methods such as the use of reporter genes. Thisobservation lead to approaches such as the co-transfection, co-selectiontechnique reported herein that involves reporter genes.

It has been discovered, however, that TALENs modification can beperformed with an efficiency that is so great that reporters are notneeded and their use merely delays the creation of transgenic animallines. Without being bound to a particular theory, a number of factorsindependently contributed to the invention of the reporter-freeembodiments. One is the realization that TALENs tend to act quickly andat a high efficiency. However, TALENs modifications tended to beunstable over a time frame of several days such that efficiencies canseem to be low depending on the time of sampling. Further, it wasconventional wisdom that only stably modified organisms should be usedto make transgenic animals so that there is little incentive tounderstand short-term modifications. There is an incentive to use cellsurvival genes to select for stable incorporation, as is conventionallydone in other systems. Another factor is that TALENs mRNA isunexpectedly effective as compared to vectors that express the TALENs.Direct introduction of mRNA encoding TALENs is, in general, useful, andwas used in Examples 12 to 17.

Another factor is that, when an HDR template is desired, directintroduction of ssDNA, e.g., single stranded (ss) oligonucleotides, isuseful, as demonstrated in Example 11. A confounding effect is that thetiming of the delivery of ssDNA was important. In Example 11, deliveryof the ss oligonucleotides at the same time as the TALENs encoded fromplasmid DNA was not effective, but delaying introduction of the ssoligonucleotides for 24 hours resulted in high efficiencies. On theother hand, Example 15 showed that simultaneous introduction of ssoligonucleotide templates and TALENs mRNA was effective. Since TALENswere introduced in Example 11 as plasmid DNA expression cassettes, theremay have been 12 or more hours of delay between transfection andaccumulation of enough TALEN protein to begin cleaving the target.Perhaps the oligonucleotides introduced with the TALENs in Example 11were degraded by the cells or otherwise un-available (compartmentalizedor in complex with proteins) to act as template for HR at the same timethat TALENs were actively cleaving the target. Another confoundingfactor, surprisingly, was that the ss nucleotides have a biphasic effect(Example 15). That is to say, too little or too much ss oligonucleotideresults in a low frequency of HDR. Embodiments of the invention includethose wherein the ssDNA is introduced into the cell after a vectorencoding a TALEN is introduced into the cell, for instance, betweenabout 8 hours and about 7 days afterwards; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., from about 1 to about 3 days hours.Embodiments of the invention include those wherein the ssDNA isintroduced into the cell at about the same time as mRNA encoding aTALENs is directly introduced, with the term “about the same time”meaning within less than about 7 hours of each other.

Another factor contributing to discovery of reporter-free embodimentswas that there is an unexpected synergy between ssDNA (ssoligonucleotide) templates and TALENs activity. For example, delivery of0.5-10 micrograms TALEN-encoding mRNAs to 500,000-750,000 cells bynucleofection followed by 3 days of culture at 30 degrees Celsiusresults in consistent levels of modification. But supplementation ofthese same conditions with 0.2-1.6 nMol of ssODN led to an increase inTALENs activity, as observed by increased NHEJ as assayed by SURVEYORassay (Example 15). Typically, a transfection consists of 1-4 microgramsof TALEN mRNA and 0.2-0.4 nMol of ssDNA. Embodiments include introducingto a cell or an embryo, an amount of TALEN mRNA that is more than about0.05 μg per 500,000 cells, or in a range of from about 0.05 μg to about100 μg per 500,000 cells; artisans will immediately appreciate that allthe ranges and values within the explicitly stated ranges arecontemplated. Embodiments include further introducing ssDNA at aconcentration of more than about 0.02 nMol or in a range of from about0.01 to about 10 nMol of ssDNA.

The term direct introduction, e.g., direct mRNA introduction, refers tointroduction of mRNA material. In contrast, introduction by means of avector encoding the mRNA is termed indirect introduction. Many processesof direct introduction are known, e.g., electroporation, transfection,lipofection, liposome, nucleofection, biolistic particles,nanoparticles, lipid transfection, electrofusion, and direct injection.

Certain embodiments of the invention are directed to processes ofmodifying cells or embryos without the use of reporters and/or selectionmarkers. In general, it was observed that TALENs and CRISPR/Cas9modifications were unstable over a time frame of several days.Accordingly, processes described herein for stabilizing changes may beused, as well as other processes described in US 2013/0117870: forinstance, direct mRNA introduction and/or use of ssDNA templates. Theterm direct introduction, e.g., direct mRNA introduction, refers tointroduction of mRNA material. In contrast, introduction by means of avector encoding the mRNA is termed indirect introduction. Many processesof direct introduction are known, e.g., electroporation, transfection,lipofection, liposome, nucleofection, biolistic particles,nanoparticles, lipid transfection, electrofusion, and direct injection.

Founder animals can be immediately created from modified cells orembryos without the need to create initially modified animals that aresubsequently bred to create the basis for a new transgenic line. Theterm founder or founder animal is used to refer to a first-generation(“F0”) transgenic animal that develops directly from the cloned cell ortreated/injected embryo that is modified. Methods reported hereinprovide for creation of founders genetically modified only at thechromosomal target site, and without intermediate steps of breedingand/or inbreeding. Moreover, embodiments include founders that arehomozygous for the modification. The founders may be prepared withoutever exposing cells and/or embryos to reporter genes (and/or selectionmarker genes).

Embodiments include a method of making a genetically modified animal,said method comprising exposing embryos or cells to an mRNA encoding aTALEN, with the TALEN specifically binding to a chromosomal target sitein the embryos or cells, cloning the cells in a surrogate mother orimplanting the embryos in a surrogate mother, with the surrogate mothergestating an animal that is genetically modified without a reporter geneand only at the chromosomal target site bound by the TALEN. The animalmay be free of all reporter genes or may be free of selection markers,e.g., is free of selection markers but has a reporter such as afluorescent protein. Options include directly introducing the TALENs asmRNA and/or a ss oligonucleotide that provides a template for a geneticmodification, e.g., an allele.

A method of making a genetically modified animal comprises introducingTALENs and/or vectors into cultured cells, e.g., primary livestockcells. The TALENs are directed to specific chromosomal sites and cause agenetic alteration at the site. An HDR template may also be introducedinto the cell, e.g., as a double stranded vector, single stranded DNA,or directly as a ss nucleotide. The cultured cells are subsequentlycultured to form colonies of clonal cells. The colonies are tested byPCR and/or sequenced, or otherwise assayed for a genetic modification,preferably without a reporter gene and/or without a selection marker.Cells are taken from colonies that are genetically modified at theintended site and used in cloning. For example, from 10 to 50,000 cellsare used to make from 10 to 50,000 embryos that are implanted intosurrogates, e.g., in sets of 1-500 embryos per surrogate; artisans willimmediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated. Embodiments comprise exposingthe cells to the TALEN without a reporter gene, creating colonies ofclonal cells, and testing a subset of members of the colonies toidentify colonies incorporating the modification at the chromosomaltarget site.

Processes of making colonies of clonal cells from cultured cells areknown. One such method involves dispersing cells from a first cultureinto a second culture wherein the various cells are not in contact witheach other, e.g., by diluting the cells into multiwall plates or into aplate with a relatively large surface area for the number of cellsplaced therein. The cells are cultured for a period of time that allowsthe cells to multiply. The multiplying cells are cultured in conditionswhere they are not likely to move far away from their original location.As a result, a user may observe the cells after the period of time andsee various colonies that are all made of a single cell and its progeny.A subset of the cells in the colony may be sampled without destroyingthe other cells in the colony.

Assays for a genetic modification may include destructive assays,meaning an assay that destroys the cell that is tested to determine ifit has a certain property. Destructive assays provide an opportunity torapidly, thoroughly, and directly test for a medication. Destructiveassays are made practical by a taking a sample of a clonal colony. Manysuch assays are highly efficient, particularly when the intendedmodification is known. For example, PCR may be performed to identifyindels or mismatches in pre-existing sequences, or to detect a sequenceof a HDR template. Or, for example, cellular DNA may be nucleolyticallyassayed, e.g., to determine if a novel nuclease target sequence has beensuccessfully introduced or knocked-out. Example 17 uses a nucleolyticdestructive assay. Other processes may be used that involve, e.g.,sequencing or SDS-PAGE to find a band that is indicative of amodification. Other processes may be used that involve, e.g., sequencingor SDS-PAGE to find a band that is indicative of a modification. Testingprocesses may be, e.g., chosen from the group consisting of anucleolytic assay, sequencing, PAGE, PCR, primer extension, orhybridization.

Allele migration has many important applications. The Allelic MigrationTable, below, describes certain genes and their applications. Artisansreading this application will be able to make and use the migrations andresultant cells and animals. Artisans can readily apply the processesset forth herein for the use of these alleles as templates or targetsfor disruption. Embodiments include making a genetically modified cellor animal (for instance, a lab animal, an F0 founder, or animal line)that has a genome with a has received a gene from the Table, e.g., byinsertion or template-driven allele migration. Alleles for some genesare reported to provide livestock production advantages, but are at verylow frequencies or are absent in some breeds or species (see items 1-9).Introgression of these alleles can be of significant value forproduction traits. For example, the Polled allele (item 1) from beefbreeds results in animals that do not have horns, whereas dairy breedsdo not have this allele so have horns and need to be dehorned as aproduction practice. Allele migration from beef breeds into horned(dairy) breeds will lead to hornless dairy cattle which is has value forboth production and animal welfare. Other examples relate to allelesthat can increase or enhance characteristics of agricultural productssuch as meat (items 4-6) and milk (items 7-8). Item 9 is useful fordisease resistance.

Many commercial and commonly used animal breeds have been carefully bredto establish desirable traits but, in the process of that breeding, haveaccumulated genetic errors that reduce their reproductive successbecause of losses in fertility or by increasing miscarriages.Deleterious alleles for some genes are present in animal populations. Asexplained elsewhere herein, the inventive techniques provide forchanging alleles only at an intended location in a target animal,without other modifications resulting from genetic tools or from meioticrecombinations. Therefore, for the first time, it is possible toclean-up the genetic errors that have accumulated in livestock andanimal breeds without disrupting the genome of the animals and,consequently, disrupting traits or causing unintended consequences.Alleles for some genes can be used to control animal fertility forgenetic control of breeding stock (items 2-3).

Many useful animal models can be made. Certain alleles are useful, seeitems 10-39. Some of these are established in animals. Others of thegenes are known to cause human disease, so introgressing these allelesinto livestock, lab animals, or other animals is useful to createbiomedical models of human disease.

Embodiments of the invention include a method of making a geneticallymodified animal, said method comprising exposing embryos or cells to anmRNA encoding a TALEN, with the TALEN specifically binding to a targetchromosomal site in the embryos or cells, cloning the cells in asurrogate mother or implanting the embryos in a surrogate mother, withthe surrogate mother thereby gestating an animal that is geneticallymodified without a reporter gene and only at the TALEN targetedchromosomal site wherein the allele is a member of the group consistingof (a) horn polled locus (b) a gene recessive for fertility defects,e.g., CWC15 and/or ApaF1 (c) genes for enhancing a livestock trait,e.g., meat production (GDF8, IGF2, SOCS2, or a combination thereof)and/or milk production (DGAT1 and/or ABCG2) (d) a gene for resistance toAfrican swine fever (P65/RELA) (e) a gene for reduction of animal size(GHRHR) (f) genes that potential tumor growth (e.g., TP53, APC, PTEN,RB1, Smad4, BUB1B, BRCA1, BRCA2, ST14 or a combination thereof) (g)human oncogenes for animal models of cancer (e.g., AKT1, EGF, EGFR,KRAS, PDGFRA/B or a combination thereof) (h) genes in animal models forhypercholesterolemia (to induce atherosclerosis, stroke, and Alzheimer'sdisease models), e.g., LDLR, ApoE, ApoB or a combination thereof (i)Inflammatory Bowel disease, e.g., NOD2 (j) spina bifida, e.g., VANGL1and/or VANGL2 (k) pulmonary hypertension, e.g., miR-145 (1) genes forcardiac defects, e.g., BMP10, SOS1, PTPN11, Nrg1, Kir6.2, GATA4, Hand2,or a combination thereof and (1) celiac disease genes, e.g., HLA-DQA1.

Movement of Alleles

Some livestock traits are related to alleles such as polymorphisms(large or small), single nucleotide polymorphisms, deletions,insertions, or other variations. For instance, a myostatin allele (an11-bp deletion) from Belgian Blue cattle causes a double-musclingphenotype. Example 6 shows, using the Belgian Blue allele, how toprecisely transfer specific alleles from one livestock breed to anotherby homology-dependent repair (HDR). Bovine fibroblasts received theallele and may readily be used to make transgenic cattle. This alleledoes not interfere with normal development and the methods taught hereinplace the allele with precision and without disruption of other genes orthe incorporation of exogenous genes. As already discussed, resultspresented herein show that the frequency of allele conversion inlivestock fibroblasts is high when sister chromatids are used for an HDRtemplate, therefore allele introgression into one sister chromatid canbe anticipated frequently to result in homozygosity.

An embodiment of this invention is a method of transfer of an allelefrom a first livestock line or breed to a second livestock line orbreed, comprising cutting DNA with a pair of TALENs in a cell or embryoof the second livestock line/breed in a presence of a nucleic acid thatencodes the allele of the first livestock line/breed. The embryo or cellmay be used to create an animal of the second line/breed that has theallele of the first line/breed. The DNA that encodes the allele providesa template for homology-dependent repair. As a template, it has homologyto portions of the DNA on each side of the cut and also contains thedesired allele.

Embodiments of the invention comprise moving a plurality of alleles fromone breed to another breed. For instance, alleles may be moved fromWagyu or Nelore cattle to Belgian Blue cattle, or vice versa. As setforth elsewhere herein, the TALENs may be delivered a protein or encodedby a nucleic acid, e.g., an mRNA or a vector. A reporter may also betransfected into the cell or embryo and used as a basis for selectingTALEN-modified cells. The reporter may be assayed non-destructivelyand/or may comprise a selection marker. Similarly, allele migration maybe practiced in an animal model.

A population or species of organisms typically includes multiple allelesat each locus among various individuals. Allelic variation at a locus ismeasurable as the number of alleles (polymorphisms) present, or theproportion of heterozygotes in the population. For example, at the genelocus for the ABO blood type carbohydrate antigens in humans, classicalgenetics recognizes three alleles, IA, IB, and IO, that determinecompatibility of blood transfusions. An allele is a term that means oneof two or more forms of a gene.

In livestock, many alleles are known to be linked to various traits suchas production traits, type traits, workability traits, and otherfunctional traits. Artisans are accustomed to monitoring and quantifyingthese traits, e.g., Visscher et al., Livestock Production Science, 40(1994) 123-137, U.S. Pat. No. 7,709,206, US 2001/0016315, US2011/0023140, and US 2005/0153317. Accordingly, the allele that istransferred may be linked to a trait or chosen from a trait in the groupconsisting of a production trait, a type trait, a workability trait, afertility trait, a mothering trait, and a disease resistance trait.

The term natural allele in the context of genetic modification means anallele found in nature in the same species of organism that is beingmodified. The term novel allele means a non-natural allele. A humanallele placed into a goat is a novel allele. The term synthetic allelemeans an allele that is not found in nature. Thus a natural allele is avariation already existing within a species that can be interbred. And anovel allele is one that does not exist within a species that can beinterbred. Movement of an allele interspecies means from one species ofanimal to another and movement intraspecies means movement betweenanimals of the same species. Moving an allele from one breed to anotherby conventional breeding processes involves swapping many allelesbetween the breeds. Recombination during meiosis inevitably exchangesgenetic loci between the breeds. In contrast, TALENs-modified livestockand other animals are free of genetic changes that result from meioticrecombination events since the cells or embryos are modified at a timewhen cells are exclusively mitotic. As a result, a TALEN-modified animalcan easily be distinguished from an animal created by sexualreproduction.

The processes herein provide for editing the genomes of existinganimals. The animal has a fixed phenotype and cloning the animal, e.g.,by somatic cell cloning, effectively preserves that phenotype. Making aspecific change or changes in a cellular genome during cloning allowsfor a known phenotype to be altered. Processes herein alternativelyprovide for altering a genome of an embryo that has yet to develop intoan animal with fixed traits. Embryos with sound genetics may nonethelessnot express all of the traits that are within the genetic potential oftheir genetics, i.e., animals do not always express the traits thattheir line is bred for. Embodiments include providing embryos havinggenetics known to be capable of expressing a set of traits and exposingthe embryos to the TALEN (optionally without a reporter gene and/orselection marker) and screening the gestated animal for the modificationand for expression of the set of traits. Accordingly, the introgressionof desirable alleles into livestock can be achieved by editing thegenomes of animals previously determined to be of significant geneticvalue by somatic cell modification and cloning, or by editing thegenomes of animals prior to determining their implicit genetic value bytreatment/injection of embryos. In the case of cloning, geneticallysuperior animals could be identified and subjected to gene editing forthe correction of a loss of function allele or the introgression ofdesirable alleles that are not already present. This approach providesfor a controlled and characterized outcome at every step of the processas only cells harboring the desired changes would be cloned.

Editing could also be applied by the direct treatment of embryos.Embryos of unknown genetic merit would be treated and screening ofoffspring may consist of analysis for the desired change and analysis ofgenetic merit of the animal, e.g., analysis for the change plus analysisof various traits that the animal expresses. A beneficial aspect of thisapproach is it can be applied simultaneous with genetic improvement bymarker assisted selection whereas cloning results in the loss of 1+generation intervals. The efficiency of such modifications would need tobe sufficiently high to offset any losses in reproductive rateengendered by embryo treatment. In the case of simple gene-inactivation,the frequency of success is very high (75%), with even homozygousmodification in 10-20% of embryos (Examples 1 and 8). Embodimentsinclude exposing embryos (or cells) to a TALEN (optionally without areporter gene and/or without a marker gene with more than about 1% ofthe embryos incorporating the modification at the TALEN-targetedchromosomal site (heterozygous or homozygous); artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., from about 1% to about 85%, or at leastabout 5% or at least about 10%. Cells may similarly have TALENsintroduced successfully at very high efficiencies, with the same rangesbeing contemplated, i.e., more than about 1%. Conventional processesachieve a lower percentage. Moreover, precision genome editing can alsobe used to introduce alleles that do not currently exist within aspecies by homology-driven allelic substitution.

Introgression of POLLED Allele

To protect the welfare of dairy farm operators and cattle, horns areroutinely manually removed from the majority of dairy cattle in theU.S., Europe, and in other regions. De-horning is painful, elicits atemporary elevation in animal stress, adds expense to animal productionand, despite the intent of protecting animals from subsequent injury,the practice is viewed by some as inhumane. Some beef breeds arenaturally horn-free (e.g., Angus), a trait referred to as POLLED that isdominant. The techniques set forth herein improve animal well-being byproviding animals that do not have to undergo dehorning. Two allelicvariants conferring polledness have recently been identified onchromosome 1. Dairy cows with either of these mutations are rare andgenerally rank much lower on the dairy genetic selection indices thantheir horned counterparts. Meiotic introgression of the POLLED alleleinto horned breeds can be accomplished by traditional crossbreeding, butthe genetic merit of crossbred animals would suffer and require manylengthy generations of selective breeding to restore to productivity.

It is possible, however, to create polledness in animals, and to do sowithout disturbing the animals' genome. The non-meiotic introgression ofthe Celtic POLLED allele (duplication of 212 bp that replaces 10 bp) wasachieved in fibroblasts derived from horned dairy bulls. A plasmid HDRtemplate containing a 1594 bp fragment including the Celtic POLLEDallele was taken from the Angus breed (FIG. 35 panel a and FIG. 72).TALENs were designed such that they could cleave the HORNED allele butleave the POLLED allele unaffected. Surprisingly, this experiment showedthat one pair of TALENs delivered as mRNA had similar activity comparedto plasmid expression cassettes (FIG. 36), Accordingly, experiments wereperformed that delivered TALENs as mRNA to eliminate the possiblegenomic integration of TALEN expression plasmids. Five of 226 colonies(2%) passed each PCR test shown in FIG. 6 panel b to confirmintrogression of POLLED. Three of the five clones were homozygous forPOLLED introgression and confirmed by sequencing to be 100% identical tothe intended allele. U.S. Ser. No. 14/154,906 filed Jan. 14, 2014, whichis hereby incorporated by reference herein, provides additionalinformation regarding polledness.

Embodiments of the invention comprise moving a polled allele from onebreed to another breed. For instance, alleles may be moved from Anguscattle to other cattle. Horned breeds include: Hereford, Shorthorn,Charolais, Limousin, Simmental, Brahman, Brangus, Wagyu, and SantaGertrudis, Ayrshire, Brown Swiss, Canadienne, Dutch Belted, Guernsey,Holstein (Holstein-Friesian), Jersey, Kerry, Milking Devon, MilkingShorthorn, Norwegian Red, Busa, Canadienne, Estonian Red, Fleckveih,Frieian, Girolando, Illawarra, Irish Moiled, Lineback, Meuse RhineIssel, Montbeliarede, Normande, Randall, Sahhiwal, Australian MilkingZebu, Simmental, Chianina Marchigiana, Romagnola. Some of the abovelisted breeds also have polled variants, but the lines in which theregenetics are often inferior to the horned version. Examples of polledbreeds include: Angus, Red Angus, Red Poll, Galloway, Belted Galloway,American White Park, British White, Amerifax, Jamaica Black, JamaicaRed, Murray Grey, Brangus, Red Brangus, Senopol. As set forth elsewhereherein, the site-specific endonuclease tools, e.g., TALENs, may bedelivered as a protein or encoded by a nucleic acid, e.g., an mRNA or avector.

Geneticists have hunted for the genetic locus of polledness for decades.In brief, polledness has been an object of intense modern research fortwenty years. See Allais-Bonnet et al. (2013) Novel Insights into theBovine Polled Phenotype and Horn Ontogenesis in Bovidae. PLoS ONE 8(5):e63512. The polled mutation was quickly mapped to bovine chromosome 1 inmany breeds, but the actual site of the genetic cause of polledness waselusive for various reasons. Quite recently, however, it was shown thatthere are at least two polled alleles (one “Celtic” and one “Friesian”)and candidate mutations were proposed for each of them. Medugorac et al.(2012) Bovine polledness—an autosomal dominant trait with allelicheterogeneity. PLoS One 7:e39477. None of these mutations were locatedin known coding or regulatory regions. Herein, the inventors show thatmaking genetic changes at comparable sites in non-polled (horned)animals can result in polled phenotypes.

Two cattle alleles for polled have been identified on chromosome 1 incattle (Medugorac, 2012). PC, Celtic origin (212 bp, 1,705,834-1,706,045bp) is duplicated (and replaces a sequence of 10 bp (1,706,051-1,706,060bp). Some breeds with this allele include Angus, Galloway, Fleckvieh,Gelbvieh and Murnau-Werdenfelser. A second polled allele of, PF, is ofFriesian origin is characterized by the following, P5ID (replace 7 bp(CGCATCA with TTCTCAGAATAG (SEQ ID NO: 177); 1,649,163-1,649,169) and80,128 bp duplication (1,909,352-1,989,480 bp P80kbID, plus five pointmutations at the positions (G1654405A, C1655463T, T1671849G, T1680646C,C1768587A). These changes are generally inherited as a fixed block. Allchromosomal coordinates are from the UMD 3.1 cattle genome build.

The inventors show herein that the bovine POLLED allele was introgressedinto horned Holstein fibroblasts. This example demonstrates that variousbreeds of dairy cattle can be created that do not have horns. And thischange can be made without disturbing other genes, or other parts of thegenome, of the animals. These processes have been developed by theinventors to achieve efficiencies that are so high that genetic changescan be made without reporters and/or without selection markers.Moreover, the processes can be used in the founder generation to makegenetically modified animals that have only the intended change at theintended site. These methods demonstrate meiosis-free intra- andinter-specific introgression of polled and hornless alleles in livestockcells, large mammals, and livestock for research, agricultural andbiomedical applications. Since the polled allele relates to thenon-development of horns, embryos modified (direct injection or bycloning) to be polled are expected to successfully gestate and result inlive births of healthy animals. Cells have been modified from a hornedallele to a polled allele and, as of the time of filing, steps have beentaken to clone animals from these cells and to generate live birthedanimals.

FIG. 61 describes experiments for determining if site-specific nucleasescould be made that bind to, and cleave, appropriate sites in bovine DNA.One of the problems was to determine if tandem repeats could be bound,bearing in mind that repeated sequences at the desired binding site canconfound targeting due to the high likelihood of intermolecularrecombination. Moreover, these bindings have to be efficient andmutually cooperate in a live cell in culture. The horned allele, inparticular, is a challenge due to the high similarity of polled alleleto the horned allele. The chosen location for TALEN binding sites wasnot obvious; the TALENs designs that were successful can cleave and bindthe horned locus but do not allow TALENs to cleave the polled allele.Discovering these designs was an important achievement in the researchof the invention. The success of this approach could not be predicted.As shown in FIG. 61, SEQ ID NOS: 146-153, the horned allele chosen asthe target had 212 residues and the polled allele had a repeat of those212 residues. The polled allele further had a 10 base pair (bp) deletionin between the repeats. Panel a) depicts the 212 bp sequence, with the10 bp that are to be deleted at the end, in between the left TALEN(marked by a solid inverted triangle) and the right TALEN (marked by asolid triangle). The TALENs pairs were thus placed on either edge of the10 bp deletion site. The TALENs pairs cleaved the horned allele in thearea of the 10 bp deletion. A homologous dependent recombination (HDR)template was used to guide insertion of the 212 residue repeat (actually202 residues since it is a repeat with a 10 bp deletion) between thelocations where the TALENs were binding. As depicted in panel a) atPolled, the Left TALEN and Right TALEN are then separated by 202residues. And recleavage of the polled allele is reduced. Various TALENswere made to determine if binding and cleavage could be reasonablyaccomplished. The table in panel b) lists some of the TALENs that weretested. Panel c) shows the test results with their effectivenessmeasured by the % NHEJ. The TALEN in the third lane, HP1.3, wassubsequently used for introgression of polled alleles.

FIG. 62 shows the research strategy and results for introgression of apolled allele into a cell with a horned allele. The Horned allele has1546 bp between PCR primers F1 and R1. In this sequence, there are 365bp between PCR primers F2 and R2. The horned allele with a 212 bpsequence represented by an arrow is in this area. The POLLED allele,bottom, has a tandem repeat of the 212 bp (shown as two arrows) with a10 bp deletion (not shown). The length between PCR primers F2 and R2 is567 bp; the 567 bp equals the 365 bp in the horned allele plus the 212bp repeat minus to 10 bp deletion. The length of the HDR template was1594 bp. Once the template sequence is introgressed into the cell'schromosome, there are 1746 bp between primers F1 and R1; the 1746 equalsthe 1546 bp of the horned allele plus 212 bp of the repeat minus to 10bp deletion. Further, a PCR product unique to the polled allele isindicated as P, in the tandem repeat area. TALENs were developed tospecifically target the HORNED allele (FIG. 61) which could be repairedby homologous recombination using the HDR template (SEQ ID NO: 381).Cells that received the TALENs and HDR template were diluted and platedas single-cells that were cultured and allowed to replicate in clonalcolonies. Members of the colonies were tested for the polled allele.Panel b shows representative images of colonies with homozygous orheterozygous introgression of POLLED. Three primer sets were used forpositive classification of candidate colonies: F1+R1, F2+R2 and F1+P(POLLED specific). Identity of the PCR products was confirmed bysequencing F1+R1 amplicons.

FIG. 63 is an example of polled conversion. The polled allele wasintrogressed into cells in a manner similar to that described for FIGS.1 and 2, except that a different HDR template was used. The template was591 bp in length:

(SEQ ID NO: 522) GAAGGCGGCACTATCTTGATGGAACTCAGTCTCATCACCTGTGAAATGAAGAGTACGTGGTACCAACTACTTTCTGAGCTCACGCACAGCTGGACGTCTGCGCCTTTCTTGTTATACTGCAGATGAAAACATTTTATCAGATGTTTGCCTAAGTATGGATTACATTTAAGATACATATTTTTCTTTCTTGTCTGAAAGTCTTTGTAGTGAGAGCAGGCTGGAATTATGTCTGGGGTGAGATAGTTTTCTTGGTAGGCTGTGAAATGAAGAGTACGTGGTACCAACTACTTTCTGAGCTCACGCACAGCTGGACGTCTGCGCCTTTCTTGTTATACTGCAGATGAAAACATTTTATCAGATGTTTGCCTAAGTATGGATTACATTTAAGATACATATTTTTCTTTCTTGTCTGAAAGTCTTTGTAGTGAGAGCAGGCTGGAATTATGTCTGGGGTGAGATAGTTTTCTTTGCTCTTTAGATCAAAACTCTCTTTTCATTTTTAAGTCTATCCCAAAAGTGTGGGAGGTGTCCTTGATGTTGAATTATAGGCAGAGGGTCAGTTTATCAACACCCAAGACCAACATCTCTGCC.

As indicated by the arrowhead, one of the 12 colonies had a PCR productthat demonstrated introgression of the polled allele.

FIG. 64 depicts another scheme for introgression of a polled allele intoa cell. A 325 bp HDR template was used:

(SEQ ID NO: 14) 5′gtctggggtgagatagttttcttggtaggctgtgaaatgaagagtacgtggtaccaactactttctgagctcacgcacagctggacgtctgcgcctttcttgttatactgcagatgaaaacattttatcagatgtttgcctaagtatggattacatttaagatacatatttttctttcttgtctgaaagtctttgtagtgagagcaggctggaattatgtctggggtgagatagttttctttgctctttagatcaaaactctcttttcatttttaagtctatcccaaaagtgtgggaggtgtccttgatgttgaattataggcag.

The introgressed allele was Red Angus polled and the recipient washorned Holstein fibroblasts. The template had 29 bp of upstream overlapand 84 bp of downstream overlap. The 212 bp repeat was in between theoverlaps. The repeat was used as a replacement for the 10 bp deletion ofthe native 212 bp sequence. This process was similar to those describedin FIGS. 1-3 except that a heat denatured (single stranded) oligomer ofTALENs was used. As shown in FIG. 64, panel's b and c, there were twoconditions tested. In panel b), the cells were transfected with 2 μg ofTALEN mRNA+500 ng of ssDNA coated with Gal4:RecA. Each lane/PCR reactionconsists of ˜3 cell equivalents diluted from a transfected population.PCR using primers btHP-F1 and btHP-R1 from horn cells results in aproduct of 389 bp. Conversion to polled results in a net insertion of202 base pairs; thus the PCR product of the same primers results in a591 bp product (arrow in left margin). The number of reactions withproducts indicative of polled conversion is shown in the upper rightcorner. Panel c) PCR assessment of polled conversion in horned Holsteinfibroblasts transfected with 2 ug of TALEN mRNA+1,500 ng of ssDNA. Thenumber of reactions with products indicative of polled conversion isshown in the upper right corner.

FIG. 65 shows allele introgression with CRISPR/Cas9. This method iscompared to a TALENs method. The introgressed allele is Adenomatouspolyposis coli (APC). At panel a) the APC14.2 TALENs and the gRNAsequence APC14.2 G1a are shown relative to the wild type APC sequence.Below, the HDR oligo is shown which delivers a 4 bp insertion (see boxedsection) resulting in a novel HindIII site. Pig fibroblasts transfectedwith 2 μM of oligo HDR template, and either 1 μg TALEN mRNA, 1 μg eachplasmid DNA encoding hCas9 and the guidance RNA (gRNA) expressionplasmid; or 1 μg mRNA encoding hCas9 and 0.5 m of gRNA expressionplasmid, were then split and cultured at either 30 or 37° C. for 3 daysbefore expansion at 37° C. until day 10. At panel b) the charts displayRFLP and Surveyor assay results. TALEN stimulated HDR was most efficientat 30° C., while CRISPR/Cas9 mediated HDR was most effective at 37° C.For this locus, TALENs were more effective than the CRISPR/Cas9 systemfor stimulation of HDR despite similar DNA cutting frequency measured bySurveyor assay. In contrast to TALENs, there was little difference inHDR when hCas9 was delivered as mRNA versus plasmid.

Control of Maturation in Animals

It is desirable to produce livestock in a way that conservesenvironmental and energy resources. Sexually immature animals generallyconsume less food per pound of weight than mature or maturing animals.Livestock, in general, do not reach a desirable size before maturation.Set forth herein, however, are animals that can be grown to a desirablesize before maturation.

In fact, methods are described herein whereby an animal does notsexually mature at all. It can be grown past the normal age of maturitywithout passing through pubescence. Sexually immature animals aresterile. The efficient production of sterile animals is therefore asignificant challenge since sexual reproduction is cost effective, andeven assisted reproductive techniques (ARTs) require a mature animal toprovide ova and sperm. In some embodiments, the livestock animal doesnot pass into puberty and remains permanently sexually immature unlessspecifically treated to allow it to pass into sexual maturity. Suchanimals, after treatment to induce maturity, can then be bred.

An advantage of making livestock incapable of maturing is that they areunable to reproduce. In the case of sexually-bred or geneticallymodified fish, for instance, concerns about their accidental releaseinto the wild are eliminated. Other animals that are similarly modifiedwill also be unable to reproduce, so that animals with valuable genetictraits can be sold without concerns of uncontrolled breeding of theanimals by the buyers. Further, in many farm animals (e.g., cows,poultry, and fish) sterilization will increase productivity as well asmeat quality, improvements in lipid content, pigmentation and texture.The term cow is a colloquial term for cattle; cattle are largeungulates, are the most widespread species of the genus Bos. And, in thecase of fish, sterile fish should demonstrate greater performance inculture by conserving energy for growth rather than gonad developmentand sexual differentiation. Currently, sterilization through ploidymanipulation (specifically triploidy, which adds of one extra set ofchromosomes) is the only commercially scalable technique available toaquaculture producers. However, inconsistent results have raisedconcerns with respect to the efficacy of the technique. In addition,triploid induction, in general, often negatively impacts survival and/orperformance of treated populations. And the application of thetechnology is labor intensive, logistically complicated and costly.

An embodiment of the invention is a genetically modified livestockanimal comprising a genome that comprises an inactivation of aneuroendocrine gene selective for sexual maturation, with theinactivation of the gene preventing the animal from becoming sexuallymature. The gene is selectively directed to sexual maturation processesand, if knocked-out of an animal, the animal will be comparable towild-type animals in terms of its development as measured by size andweight until such time as the wild type animals undergo sexualmaturation. The term gene means a locatable region of genomic sequence,corresponding to a unit of inheritance, which is associated withregulatory regions, transcribed regions, and or other functionalsequence regions. The term gene, as used herein, includes the functionalsequence regions as well as those portions that encode a protein orother factor. The term knocked-out, as used herein, refers to the director indirect disruption of a gene that either inactivates function in theresulting protein or eliminates production of the protein product.

Since the genetic modifications are directed to a specific gene or geneproduct to prevent sexual maturation, the factor that is needed formaturation is known and can generally be supplied.

Inducible Systems

An inducible system may be used to control expression of a sexualmaturation gene. Various inducible systems are known that allowspatiotemporal control of expression of a gene. Several have been provento be functional in vivo in transgenic animals.

An example of an inducible system is the tetracycline (tet)-on promotersystem, which can be used to regulate transcription of the nucleic acid.In this system, a mutated Tet repressor (TetR) is fused to theactivation domain of herpes simplex virus VP16 trans-activator proteinto create a tetracycline-controlled transcriptional activator (tTA),which is regulated by tet or doxycycline (dox). In the absence ofantibiotic, transcription is minimal, while in the presence of tet ordox, transcription is induced. Alternative inducible systems include theecdysone or rapamycin systems. Ecdysone is an insect molting hormonewhose production is controlled by a heterodimer of the ecdysone receptorand the product of the ultraspiracle gene (USP). Expression is inducedby treatment with ecdysone or an analog of ecdysone such as muristeroneA. The agent that is administered to the animal to trigger the induciblesystem is referred to as an induction agent.

The tetracycline-inducible system and the Cre/loxP recombinase system(either constitutive or inducible) are among the more commonly usedinducible systems. The tetracycline-inducible system involves atetracycline-controlled transactivator (tTA)/reverse tTA (rtTA). Amethod to use these systems in vivo involves generating two lines ofgenetically modified animals. One animal line expresses the activator(tTA, rtTA, or Cre recombinase) under the control of a selectedpromoter. Another set of transgenic animals express the acceptor, inwhich the expression of the gene of interest (or the gene to bemodified) is under the control of the target sequence for the tTA/rtTAtransactivators (or is flanked by loxP sequences). Mating the twostrains of mice provides control of gene expression.

The tetracycline-dependent regulatory systems (tet systems) rely on twocomponents, i.e., a tetracycline-controlled transactivator (tTA or rtTA)and a tTA/rtTA-dependent promoter that controls expression of adownstream cDNA, in a tetracycline-dependent manner. In the absence oftetracycline or its derivatives (such as doxycycline), tTA binds to tetOsequences, allowing transcriptional activation of the tTA-dependentpromoter. However, in the presence of doxycycline, tTA cannot interactwith its target and transcription does not occur. The tet system thatuses tTA is termed tet-OFF, because tetracycline or doxycycline allowstranscriptional down-regulation. Administration of tetracycline or itsderivatives allows temporal control of transgene expression in vivo.rtTA is a variant of tTA that is not functional in the absence ofdoxycycline but requires the presence of the ligand for transactivation.This tet system is therefore termed tet-ON. The tet systems have beenused in vivo for the inducible expression of several transgenes,encoding, e.g., reporter genes, oncogenes, or proteins involved in asignaling cascade.

The Cre/lox system uses the Cre recombinase, which catalyzessite-specific recombination by crossover between two distant Crerecognition sequences, i.e., loxP sites. A DNA sequence introducedbetween the two loxP sequences (termed floxed DNA) is excised byCre-mediated recombination. Control of Cre expression in a transgenicanimal, using either spatial control (with a tissue- or cell-specificpromoter) or temporal control (with an inducible system), results incontrol of DNA excision between the two loxP sites. One application isfor conditional gene inactivation (conditional knockout). Anotherapproach is for protein over-expression, wherein a foxed stop codon isinserted between the promoter sequence and the DNA of interest.Genetically modified animals do not express the transgene until Cre isexpressed, leading to excision of the foxed stop codon. This system hasbeen applied to tissue-specific oncogenesis and controlled antigenereceptor expression in B lymphocytes. Inducible Cre recombinases havealso been developed. The inducible Cre recombinase is activated only byadministration of an exogenous ligand. The inducible Cre recombinasesare fusion proteins containing the original Cre recombinase and aspecific ligand-binding domain. The functional activity of the Crerecombinase is dependent on an external ligand that is able to bind tothis specific domain in the fusion protein.

Embodiments include an in vitro cell, an in vivo cell, and a geneticallymodified animal such as a livestock animal that comprise aneuroendocrine gene selective for sexual maturation that is undercontrol of an inducible system. The genetic modification of an animalmay be genomic or mosaic. An embodiment is a gene in the groupconsisting of Gpr54, Kiss1, and GnRH1 that is under control of aninducible system. The inducible system may be, for instance, selectedfrom the group consisting of Tet-On, Tet-Off, Cre-lox, and Hif1alpha.

Neuroendocrine Genes Selective for Sexual Maturation

Sexual development of animals may be prevented by blockingneuroendocrine genes selective for sexual maturation. Sexualdevelopment, accelerated growth, and adrenal maturation is initiatedwhen gonadotropin-releasing hormone (GnRH1) begins to be secreted by thehypothalamus. The gene GnRH1 encodes the GnRH11 precursor. In mammals,the linear decapeptide end-product is generally synthesized from a92-amino acid preprohormone. Gonadotropin-releasing hormone (GnRH1),also known as Luteinizing-hormone-releasing hormone (LHRH) andluliberin, is responsible for the release of follicle-stimulatinghormone (FSH) and luteinizing hormone (LH). GnRH1 belongs togonadotropin-releasing hormone family. Embodiments of the inventioninclude inactivating GnRH1 in a livestock animal. Gonadotropin-releasinghormone or analogues may be administered to the animal to bring it tosexual maturity. Sequences for GnRH1 across multiple species are wellknown, e.g., Gene IDs 768325 for Bos Taurus, 770134 for Gallus gallus,or 397516 for Sus scrofa. GPR54, also known as the Kisspeptin receptor(also referred to as GpR54, KissR, Kiss1R, kissR and the like), binds tothe hormone Kisspeptin (formerly known as metastin). Kisspeptin is aproduct derived from the KiSS1 gene (also referred to as Kiss, Kiss1,KiSS, kiss1 and the like). Kisspeptin-GPR54 signaling has a role ininitiating secretion of GnRH1. Kisspeptin is an RFamide neuropeptidewith multiple functions, involving varied whole body physiologicalsystems and acting at all levels of the reproductive axis—brain,pituitary, gonad (BPG), and accessory organs. Kisspeptin can directlystimulate GnRH release (Messager et al., 2005), relaying steroid hormonenegative and positive feedback signals to GnRH neurons, serving as agatekeeper to the onset of puberty, and relaying photoperiodicinformation.

Embodiments of the invention include inactivating the gene GPR54 and/orKiSS1 in a livestock animal. Kisspeptin may be administered to make-upfor a loss of KiSS1 and thereby achieve sexual maturity. Or, KiSS1and/or GPR54 is suppressed, and gonadotropin-releasing hormone may beadministered to the animal to bring it to sexual maturity. Anotherembodiment is inactivation of the Kisspeptin-GPR54 interaction byinserting a dominant negative GPR54 into the genome of a livestockanimal. Expression of the dominant negative GPR54 prevents initiation ofsexual maturation. Expression of the dominant negative GPR54 interfereswith signal transduction downstream of the receptor, preventing signalpropagation and release of GnRH1. Sequences for GPR54 are well knownacross multiple species, e.g., 84634 for Homo sapiens, 561898 for Daniorerio, or 733704 for Sus scrofa. Sequences for Kiss1 are well knownacross multiple species, e.g., 615613 for Bos taurus, 733704 for Susscrofa, or 100294562 for Ovis aries.

The Gpr54/Kiss pathway is highly conserved among most vertebrate speciesand is known to be the gatekeeper to puberty in humans and mice.(Seminara et al., 2003). Infertility due to inactivation of the Gpr54and/or Kiss gene in humans and mice has been reverted by ectopic GnRHadministration. Studies in mice and humans demonstrate that inactivationGpr54 effectively leads to infertility of both sexes due tohypogonadotropic hypogonadism (d'Anglemont de Tassigny et al., 2007; deRoux et al., 2003). The Kiss-Gpr54 system is highly conserved invertebrates (Tena-Sempere et al., 2012) particularly in mammals whereonly one Kiss and Gpr54 gene is present. Whereas multiple distinct Kissgenes have been identified in fish, the receptor Gpr54 is encoded by onegene in all but one species examined. Humans and mice with Gpr54mutations displayed normal levels of hypothalamic GnRH suggestingKiss/Gpr54 signaling was responsible for the release of GnRH into theblood stream (Seminara et al., 2003). This presented an opportunity tobypass Kiss/Gpr54 signaling by injection of GnRH or gonadotropinsdirectly into Gpr54-deficient subjects. Indeed, both Gpr54-deficienthumans and were responsive to GnRH injection (Seminara et al., 2003)indicating that downstream signaling components of puberty remainintact.

Direct evidence of a piscine kisspeptin role in reproductive biologypublications is lacking or limited. However, administration of kisspeptide has been shown to stimulate gonadotropin gene expression in thepituitary of sexually mature female zebrafish (Kitahashi et al. 2008)and orange grouper, or secretion of LH and FSH in European sea bass(Felip et al., 2008) and goldfish. Thus, in theory, the fertility ofsexually immature and sterile fish with knockouts of GPR54 and/or KiSS1can be rescued by exogenous delivery of kisspeptin analogues (e.g.,Kisspeptin 10) or gonadotropin analogues (LH or FSH). With this concept,homozygous kiss or kiss receptor knockout-broodstock can be bred incaptivity if administered the corrective hormone, ensuring reversiblecontrol over fertility. The progeny from this KO-broodstock inherits thealteration. This would provide economic and environmental benefit.

Neuroendocrine genes selective for sexual maturation can be inactivatedby a number of processes. Inactivation of the gene prevents expressionof a functional factor encoded by the gene, such as a protein or an RNA.One kind of inactivation comprises an insertion, deletion, orsubstitution of one or more bases in a sequence encoding the sexualmaturation factor and/or a promoter and/or an operator that is necessaryfor expression of the factor in the animal. The inactivation may be aknock-out of a gene. The gene may be inactivated by removal of at leasta portion of the gene from a genome of the animal, alteration of thegene to prevent expression of a functional factor encoded by the gene,an interfering RNA (expressed by a gene in a genome of the animal or ina plurality of cells of the animal), or expression of a dominantnegative factor by an exogenous gene.

Another system for revertible-infertility is Tac3/TacR3 (Young, J.,Bouligand, J., Francou, B., Raffin-Sanson, M. L., Gaillez, S.,Jeanpierre, M., Grynberg, M., Kamenicky, P., Chanson, P.,Brailly-Tabard, S., et al. (2010). TAC3 and TACR3 defects causehypothalamic congenital hypogonadotropic hypogonadism in humans. J ClinEndocrinol Metab 95, 2287-2295. As with Kiss/Gpr54, humans deficient forthese genes display hypogonadotropic hypogonadism which was revertibleby pulsatile GnRH treatment (Young et al., 2010). Tac and/or Tac3 may beinactivated using methods described or referenced herein.

Embodiments of the invention include methods of inactivating one or moregenes selected from the group consisting of GnRH1, GPR54, KiSS1, Tac andTac3 in animals selected from the group consisting of cattle, sheep,pigs, chickens, turkeys, goats, sheep, fish, buffalo, emu, rabbits,ungulates, avians, rodents, and livestock. The genes may be inactivatedin cells and/or embryos and in animals resulting therefrom. Variousmethods are described herein, e.g., knocking out a gene in a cell orembryo using TALENs or Zinc Finger Nucleases, and cloning and/orimplanting the cell/embryo in a surrogate to make a founder animal.

FIG. 49 illustrates an embodiment of the invention, with bovine cellsbeing modified in vitro and used to clone calves. The calves may beraised to a suitable weight for slaughter or treated with factors thatallow them to pass into maturity, e.g., gonadotropin analogues or afactor that directly supplied a knocked-out genetic factor.

Example 20 describes techniques for making changes to cells with a TALENsystem. Example 21 describes the dilution cloning technique used for theresults of Table 7 (SEQ ID NOS: 328-335 and 464, 465). Example 22describes the techniques of mutation detection and RFLP analysis.Example 41 (FIG. 50) describes introgression of an 11-basepair deletioninto exon-3 of bovine GDF8 (Belgium Blue mutation)(SEQ ID NOS:428 and431). FIG. 51 depicts results for a similar process that introgressed anallele from one species into another species. Example 42 describestesting for the same as well as introgression of alleles into cow cellsusing oligo HDR. In Example 42, TALEN-induced homologous recombinationeliminated the need for linked selection markers. When transfectedalone, the btGDF8.1 TALEN pair (SEQ ID NOS: 428 and 431) cleaved up to16% of chromosomes at the target locus. Co-transfection with asupercoiled homologous DNA repair template harboring the 11 bp deletionresulted in a gene conversion frequency (HDR) of 0.5 to 5% at day 3without selection for the desired event. Gene conversion was identifiedin 1.4% of isolated colonies that were screened by PCR, which was arapid method to identify successful alterations. Example 43 (FIG. 52)describes the modification of four intended loci in pig and cattlefibroblasts The TALEN pairs used were ssILRG2.1 (SEQ ID NOS: 484 and485); ssRAG2.1 (SEQ ID NOS:488 and 489); btGGTA9.1 (SEQ ID NOS:490 and491); and ssLDLR2.1 (SEQ ID NOS:438 and 439). Example 44 (FIG. 53) showsanalysis of modifications made to genes APC (SEQ ID NOS:482 and 483),LDLR (SEQ ID NOS:438 and 439), p53 (SEQ ID NOS:452 and 453), p65 (SEQ IDNOS:440 and 441), and btGDF8 (SEQ ID NOS:428 and 431). Example 45 (Table7) shows a recovery rate for intended indels of 10-64% (average, 45%),with up to 32% of the colonies homozygous for the modification. Example46 (FIG. 54) describes cloned pigs that were made with modified deletedin azoospermia-like (DAZL, SEQ ID NOS: 182-183) and adenomatouspolyposis coli (APC, SEQ ID NOS:188-189) genes. Example 47 (FIG. 55)describes GPR54 (SEQ ID NO: 196) knockouts, made according to theindicated gene targeting strategy; Example 48 details methods for makingmodified animals with the GPR54 knockout. Example 49 describesmodifications made with custom-made CRISPR/Cas9 endonucleases.

These results demonstrated techniques that effectively makemodifications at an intended gene, without the aid of a linked selectionmarker. Cells with the modifications can be used for cloning animals.The intended genetic modifications can be controlled with specificity,for instance, for introgressing an allele or to modify a gene.Modifications may be, for instance, a deletion or an insertion todisrupt a gene or knock it out, or to replace part of the gene to make anonfunctional gene product or an alternative product.

Fish (tilapia) with a knockout of KiSS1 and GpR54 (also referred to asGPR54, Kiss-receptor, KissR, Kiss1R) have been made. FIGS. 56 and 57depict the targeted regions for KISS and GpR54. The structuralorganization of the Kiss gene is conserved and contains two codingexons, one encoding both the signal peptide and part of the kisspeptinprecursor, the other encoding the remainder of the precursor includingthe kisspeptin-10 sequence. Example 51 details the steps that were usedto make founder fish with Kiss or KissR knockouts. Techniques based onTALENs were used to knock out the genes and melt analysis was used todetect indels (FIG. 58). Various modifications at the targeted geneswere confirmed (FIG. 59), including nine different nucleotide deletions,two insertions, and three combinations of nucleotide insertions anddeletions. Sequencing indicated that a knockout would result from atleast some of these modifications. Germ line mutations were confirmed(see FIG. 60). F1 heterozygous mutants with a Kiss or KissR knockoutwere created and bred. F2 generations, which are expected to show theinactivation phenotype, are presently being grown.

Disclosed herein are processes to make transgenic animals that havechanges only at an intended site. Additionally, the processes can makespecifically intended changes at the intended site. It is not necessaryto remove other changes resulting from problems like the use oflinked-reporter genes, or linked positive and negative selection genes,or random transgene integration are bypassed. Moreover, the processescan be used in the founder generation to make genetically modifiedanimals that have only the intended change at the intended site. Otherprocesses are also disclosed that involve unlinked marker genes and thelike.

Compositions and Kits

The present invention also provides compositions and kits containing,for example, nucleic acid molecules encoding TALENs, TALEN polypeptides,compositions containing such nucleic acid molecules or polypeptides, orTALEN engineered cell lines. Such items can be used, for example, asresearch tools, or therapeutically.

The present invention also provides compositions and kits containing,for example, nucleic acid molecules encoding site-specificendonucleases, CRISPR, Cas9, ZNFs, TALENs, polypeptides of the same,compositions containing such nucleic acid molecules or polypeptides, orengineered cell lines. An HDR may also be provided that is effective forintrogression of a polled allele. Such items can be used, for example,as research tools, or therapeutically

Example 1: Genetically Modified Artiodactyl Livestock (Bovine) Producedby Direct Injection of TALENs

Three TALEN pairs were designed and assembled as described in Cermak et.al. (2011) Nuc. Acids Res. 39:e82 (FIG. 3) and mRNA was injected intothe cytoplasm of bovine embryos at about 19 hours post fertilization.The ACAN gene was targeted. Aggrecan Proteoglycan (ACAN) is anextracellular matrix protein that can be found in cartilagenous tissues.Mutations of the ACAN gene are known to cause dwarfism in both dogs andcattle. More particularly, bulldog dwarfism in Dexter cattle is causedby mutations in ACAN. Bulldog dwarfism in Dexter cattle is one of theearliest single-locus disorders described in animals. Affected fetusesdisplay extreme disproportionate dwarfism, reflecting abnormal cartilagedevelopment (chondrodysplasia). Typically, they die around the seventhmonth of gestation, precipitating a natural abortion. Heterozygotes showa milder form of dwarfism, most noticeably having shorter legs.Homozygosity mapping in candidate regions in a small Dexter pedigreesuggested aggrecan (ACAN) as the most likely candidate gene. Mutationscreening revealed a 4-bp insertion in exon 11 (2266_2267insGGCA)(called BD1 for diagnostic testing) and a second, rarer transition inexon 1 (−198C>T) (called BD2) that cosegregate with the disorder. Inchondrocytes from cattle heterozygous for the insertion, mutant mRNA issubject to nonsense-mediated decay, showing only 8% of normalexpression. Genotyping in Dexter families throughout the world shows aone-to-one correspondence between genotype and phenotype at this locus.The heterozygous and homozygous-affected Dexter cattle could proveinvaluable as a model for human disorders caused by mutations in ACAN.

Injected embryos were cultured in vitro and collected at the blastocystsstage (FIG. 3). Individual blastocyst genomic DNA was amplified by wholegenome amplification (WGA) (Carlson et al. (2011) Trangenic Res. 20:29)and screened for indels using the SURVEYOR nuclease assay (FIGS. 3, 4A,and 4B). Cleavage products indicative of TALEN activity were observed in10% of injected embryos (e.g., FIGS. 3, 4A, and 4B). Mutations in thepredicted region were confirmed in 2 blastocysts injected with eitherACAN11 (SEQ ID NO: 377) or ACAN12 (SEQ ID NO: 378) TALEN pairs (FIG. 3).ACAN exon 11 from Bos taurus breed Hereford chromosome 21 genomicscaffold targeted by btau_4.2 TALEN pairs and ACAN exon 12 from Bostaurus breed Hereford chromosome 21 genomic scaffold targeted bybtau_4.2 were chosen. These mutations are loss of function alleles, andthus our hypothesis was that TALEN mediated KO of ACAN by targetingexons 11 (ACAN11) or 12 (ACAN12) would cause a profound and easilyobserved phenotype in mutant offspring. A significant decrease in thedevelopmental competence of TALEN-injected embryos was not observed. Asecond round of injections was then performed using the ACAN12 TALENpair at mRNA dosages ranging from 10-100 ng/μl. Comparison of theblastocyst formation rate between rounds 1 (33%) and 2 (5%) (10 ng/μlconditions) revealed poor embryo quality. Despite the poor quality ofthe embryos, 12 putative mutants (27% of injected) were identified usingthe SURVEYOR assay. The genotypes of each SURVEYOR positive embryo wereanalyzed with 14 sequencing reads from cloned PCR products. Sequencingrevealed mosaicism in gene modification. Indels were identified in 4SURVEYOR positive embryos (SEQ IDS: 2-5) and of these, indel positivesequence reads accounted for 7-29% of the total reads for each embryo,FIG. 6 and SEQ ID NOS: 28-36. Processes for the creation of animalfounder lines based on embryo transfer are well known. TALEN treatedembryos were successfully transferred to surrogate cows to establishpregnancies. These results demonstrated that TALENs functioned inartiodactyl embryos. Gene modification of embryos with Zinc FingerNucleases (ZFNs) and TALENs has been reported for model organisms bydirect injection of ZFN or TALEN mRNAs encoding a nuclease pair Geurtset al. (2009) Science 325:433; Carbery et al., (2010) Genetics 186:451;Mashimo et al. (2010) PLoS One 5:e8870; Tesson et al. (2011) NatureBiotechnol. 29:695. The sequence of the ACAN12 gene is found in FIG. 6(SEQ ID NO: 26).

Example 2: Genetically Modified Artiodactyl Livestock Produced byGenetic Modification of Bovine and Swine Somatic Cells

Several additional TALEN pairs were assembled as described in Cermark etal. (2011) Nuc. Acids Res. 39:e82 for targets in pigs and cattle chosenbased on either biomedical or agricultural relevance, such as DMD.Mammalian animal models have proven invaluable in defining thecomplexity of muscle disease and have enabled the development of severalpromising therapeutic strategies for Duchenne Muscular Dystrophy (DMD).However, the development of regenerative therapies would greatly benefitfrom more faithful and reproducible models. Muscle degeneration in themdx mouse model is mild in comparison to DMD patients, perhaps due tosmaller muscle forces in rodents, or because of partial functionalredundancy. Several dystrophin-deficient dogs have been identified andthe causative genetic lesion defined in at least three. Thebest-characterized and closest in phenotype to DMD is the goldenretriever muscular dystrophy (GRMD) dog caused by a splice-site mutationin Exon 6. However, the phenotype of the GRMD varies significantly withage and genetic background, confounding the utility of the dog model.

Pigs represent a socially and scientifically preferred large animal formodeling DMD. The musculoskeletal and cardiovascular physiology,myogenic program, and size of pigs is striking in its similarity tohumans. Like mice, the pig genome can be efficiently manipulated tocreate hypomorphic and null alleles by genome engineering. Wehypothesized that TALEN mediated mutation in porcine exons 7 or 8(DMDE6; DMDE7) would produce a reliable swine mode of DMD.

TALEN pairs DMDE6 to target DMD exon 6 (SEQ ID NO: 379) and DMDE7.1 totarget DMD exon 7 (SEQ ID NO: 380) were chosen because a high percentageof Duchene's Muscular Dystrophy (DMD) is caused by gross deletions,providing the opportunity to mimic the human condition in a porcinemodel. Binding domains of six TALEN pairs were placed in the context oftwo TALEN scaffolds (+231, Christian et. al. 2010 (op cit) and Carlson+63, see Miller et. al. 2011 (op cit)) (FIG. 5). Each TALEN pair wastransfected via plamid into primary livestock fibroblasts, and genomemodification was measured at day 3 by the SURVEYOR assay (Guschin, etal. (2010) Methods Mol. Biol. 649:247. TALEN pair sequences are listedin Table 13 and Table 14. The most active TALEN pairs, DMDE7.1 andACAN12, displayed cleavage of 38% and 25% of chromosomes, respectively,and Sanger sequencing revealed an assortment of indels characteristic ofNHEJ mediated DNA repair (FIG. 5, FIG. 6, FIG. 9, and SEQ ID NOS:56-58). The TALENs scaffold had a significant effect on activity infibroblasts. In total, 4 of 6 loci targeted with the +63 isoform cleavedat 3.5% or greater while only the DMDE7.1 TALEN pair cleaved above 1% inthe +231 scaffold (FIG. 5). As noted in Doyon et al. (2010) NatureMethods 8:74 and Miller, (2011) op. cit.), a 72 hour incubation at 30°C. after transfection also had a positive effect on activity, and wasrequired for activity of 3 TALEN pairs. The success rate for generatingactive Carlson +63 TALEN pairs has been high. Data collected shows that23 of 36 (64%) TALEN pairs were detectably active (>1.0% NHEJ) at 15genes scattered across the pig and cow genome, on autosomes and both theX and Y chromosomes. Three quarters of the active pairs cleaved withhigh efficiency (19-40%) with an average modification level of 25%.Clonal processes for the creation of animal founder lines based onmodified fibroblasts are well known. The sequence of the DIVIDE gene isfound in FIG. 9A (SEQ ID NO: 55).

Example 3: Extended Culture and Indel Enrichment by TransposonCo-Transfection

TALEN pairs were transfected into fibroblasts and cultured cells for 14+days with or without transposon co-selection prior to measurement ofmodification levels. The targeted genes include bovine GDF8 (btGDF8),Bovine ACAN (ACAN12), Porcine DMD (DMDE7.1 (A); DMDE6 (TALENs targetedto exon 6 of the DMD gene.)), Porcine LDLR (C) (LDLR2.1) (see Tables 13and 14). The results are summarized in FIG. 7, panel C. At day zero(D0), cells are transfected with a mixture of plasmids including anexpression cassette for each TALEN (two plasmids), a transposon encodinga selection marker (a third plasmid, encoding puromycin, and atransposase-expression cassette (fourth plasmid). The TALEN plasmids arethe main component (4-fold excess by mass) of each transfection.Transfected cells are cultured for 3 days at either 30 or 37° C. priorto splitting, collection of a sample for SURVEYOR assay and re-platingfor extended culture +/− selection for transposon integration. All cellsare cultured at 37° C. after day 3. Cells cultured for 14+ days arecollected for SURVEYOR assay and cryopreserved for downstreamapplications, i.e., SCNT. For comparison, other fibroblasts weretransfected by nucleofection and percent NHEJ was measured at day 3, andin day 14+ non-selected (NS) and selected (S) populations. Forcomparison, fibroblasts were also transfected using cationic-lipids.

TABLE 2 TALEN activity in bovine zygotes SURVEYOR Candidates/ TargetTrial Scaffold mRNA total ng/ul Number Inj. Blast rate assayed NHEJConfirmed Non inj. 1 — — 60 41% — — Buffer 1 — — 68 36% — — ACAN11 1 23110 67 22% 2/24 1/2 ACAN11 1 231  2 87 28% 1/32 0/1 ACAN12 1 231 10 5733% 0/22 — ACAN12 1 231  2 54 37% 1/23 1/1 PRNP3.2 1 231 10 65 14% 0/19— PRNP3.2 1 231  2 50 30% 0/17 — Subtotal- 380 4 (3%)  2/4 (1.5%assayed) ACAN12 2 231 10 59 5% 1/10 0/1 ACAN12 2 231 25 58 16% 3/16 2/3ACAN12 2 231 50 59 2% 2/9  1/2 ACAN12 2 231 100  51 0% 1/10  1/1^(a)Subtotal- 227 7 (16%) 4/7 (9% assayed) Non inj. 3 — — 51 43% — — Buffer3 — — 35 23% — — GDF83.1 3 GT   2^(b) 62 24% —  6/14 GDF83.1 3 GT 10^(b) 53 8% —  3/4^(C) Subtotal- 328  9/18 (50% assayed) ^(a)3 indelsin one embryo ^(b)eGFP mRNA was added to a final concentration of 2ng/ul. ^(c)two bi-allelic modification ACAN - Aggrecan, candidate formodel of congenital achondroplasia. PRNP - Major prion protein,implicated in spongiform encephalopathy. GDF8 - Growth differentiationfactor 8 (myostatin), regulator of muscle growth.

Example 4: Isolation of Mono- and Bi-Allelic KO Clones

Transgenic primary fibroblasts can be effectively expanded and isolatedas colonies when plated with non-transgenic fibroblasts (feeder-cells)at standard densities (>150 cells/cm2) and subjected to drug selectionusing the transposon co-selection technique applied above (Carlson etal. (2011) Transgenic Res. 20:1125). To evaluate this approach,puromycin-resistant colonies for cells treated with six TALEN pairs wereisolated and their genotypes evaluated by SURVEYOR assay or directsequencing of PCR products spanning the target site (FIGS. 8A and 8B).In general, the proportion of indel positive clones was similar topredictions made based on day 3 modification levels. Bi-alleic knockoutclones were identified for 5 of 7 different TALEN pairs, occurring in upto 35 percent of indel positive cells (Table 1). In the majority ofexamples, (15 of 23), indels were homozygous (same indel on each allele)rather than unique indels on each allele suggesting that sisterchromatid-templated repair is common (FIG. 9). Notably, among modifiedclones, the frequency of bi-alleic modification (17-60 OR35%) for themajority of TALEN pairs exceed predictions based on day 3 modification(10-17 OR15.6%) if chromosome cleavages are treated as independentevents. The sequence of the LDLR gene is found in FIG. 9B (SEQ ID NO:68). Examples of bi-alleleic sequences are further shown in FIGS. 8 and9 using LDLR2.1 and DMDE7.1 TALENs. The sequences corresponding to theTALENs listed in table 1 can be found in Tables 13 and 14.

TABLE 1 Transposon co-selection enables isolation of modified coloniesGenotype distribution in fibroblast clones. Predicted Predicted % Day 3% Mod Bi-allelic Observed Mod Observed Bi- TALEN pair Mod Clones ModClones (%) allelic Mod (%) LDLRE2.1 Pig ♂ 19 34.5 10.5 30/81 (37)  5/26(19) LDLRE2.1 Pig ♀ 21.5 38.3 12 23/76 (30)  8/23 (35)† LDLRE2.1 Pig ♂14.4 26.7 7.7 12/94 (13)  2/12 (≥17)^(A) LDLRE2.1-2x^(B) Pig 19.7 35.510.9  8/24 (33) 2/8/(≥25)^(A) LDLRE4.2 Pig ♂ 20 36 11.1  4/48 (8.3)1/4/(25)^(A) LDLRE4.2 Pig ♀ 19 34.4 10  8/47 (17)  0/8^(A) DMDE6 Pig 2543.8 15.6 17/35 (49) NA DMDE7.1 Pig 27 47 15.6 12/29 (41)  3/10 (30)DMDE7.1-2x^(B) Pig 22 39.2 12.4 22/41 (54)  7/22 (≥32)^(A)† GHRHR2.3 Pig29 50 17 26/43 (60) 15/26 (≥58)^(C)† ACAN12 Cow 29 50 17 27/35 (77)  2/6(NA)^(D) btGDF83.1 Cow 17 31 9.3 7/24/(29) 0/7 ^(A)Bi-allelic KO wereidentified by sequencing of PCR products. Only overlapping or homozygousdeletions can be identified using this technique. ^(B)Fibroblasts weretransfected and recovered twice within two weeks with the same TALENpair. ^(C)5/15 Bi-allelic colonies were confirmed as double frame-shiftalleles. ^(D)Only colonies with distinguishable gross deletions in thePCR amplicon were analyzed. †95% Confidence interval exceeds expectedbi-allelic null hypothesis

Example 5: Chromosomal Deletions and Inversions with TALENs

It was hypothesized that simultaneous delivery of two TALEN pairstargeting the same chromosome could induce large chromosomal deletions.These were achieved and, further, large inversions were incidentallydiscovered. The TALEN pairs, DMDE6 (SEQ ID NOS:434 and 437) and DMDE7.1(SEQ ID NOS:408-411) were tested because of their high activity and thefact that a high percentage of Duchene's Muscular Dystrophy is caused bygross deletions (Blake, 2002) such that a porcine model would match tothe human condition. The results are summarized in FIG. 10. Day 3 genemodification levels were high for each TALEN pair (24% for DMDE6 and 23%DMDE7.1), albeit slightly lower that when either TALEN pair wastransfected individually (FIG. 10). To determine if the sequence betweenthe two TALEN pairs had been deleted, PCR was attempted with primersspanning the TALEN target sites. If the 6.5 kb sequence had beenremoved, a band of ˜500 bp was expected, whereas the wild type band of 7kb would not be amplified with the PCR conditions chosen. A band near500 bp was observed in replicates where both TALEN pairs wereintroduced, but was absent when either TALEN pair was introduced alone(FIG. 10). The sequence of the DMD gene is found in FIG. 9A (SEQ ID NO:61).

Next, the cell population was assayed for inversion events by PCR acrosspresumptive new 5′ and 3′ junctions. Products were observed at theexpected size for both the 5′ and 3′ junctions of the presumptiveinversion only when both TALEN pairs were introduced (FIG. 10). Tovalidate further that the inversions, colonies were generated using thetransposon co-selection strategy and screened for deletion and inversionevents. Both deletion and inversion events were recovered with highfrequency (10.3% and 4.1% respectively; n>1000) (Table S4). Deletion andinversion events occurred with remarkable fidelity. Forty one out of 43of the inversion positive colonies were positive at both the 5′ and 3′junctions. Finally, sequencing of PCR products confirmed both deletionand inversion events with addition or deletion of very few nucleotidesat their junctions (FIG. 11, 12).

Example 6: TALEN-Induced Homologous Recombination Eliminates Need forLinked Selection Markers

A mutant myostatin allele (an 11 bp deletion) from Belgian Blue cattlewas placed into the genome of wild-type Wagyu cattle (Grobet et al.(1997) Nature Genet. 17:71) (FIG. 13). When transfected alone, thebtGDF8.1 TALEN pair (SEQ ID NOS: 428 and 431) cleaved up to 16% ofchromosomes at the target locus (FIG. 13). TALENs (btGDF83.1, SEQ IDNOS:428 and 431) and a dsDNA template (BB-HDR) (SEQ ID NO: 504) weredesigned to introduce an 11-bp deletion in exon-3 of bovine GDF8(Belgium Blue mutation) by DSB induced homologous recombination. Half ofthe binding site for the left TALEN was missing in the BB-HDR template,to make it resistant to TALEN cleavage. A SURVEYOR assay demonstratedactivity of btGDF83.1 TALENs at both 37 and 30° C. The PCR product usedfor this assay was generated using primers b and b′ (shown panel A ofFIG. 13). The BB-HDR template was not included in these replicates sinceit would confound estimates of btGDF83.1 activity. Allele specific PCRdemonstrated that HDR induction was dependent on co-transfection ofTALENs and the BB-HDR template. The PCR assay was developed tospecifically detect HDR modified GDF8 alleles using primers c and c′(shown panel a of FIG. 13). The 3′ end of primer c′ spanned the 11-bpdeletion, and cannot amplify the wild type allele “wt”. Five hundredcell equivalents were included in each PCR reaction including thepositive control “C”. Percent HDR was determined by comparativedensitometry between experimental and control reactions. Co-transfectionwith a supercoiled DNA template harboring a 1623 bp DNA fragment fromBelgian Blue cattle resulted in a gene conversion frequency (HDR) of0.5% to 5% as suggested by semi-quantitative PCR at day 3, withoutselection for the desired event (FIG. 13). These results demonstratedthat TALENs can be used to effectively place exogenous nucleic acidsequences in livestock, including alleles—and without markers. To assessthe frequency of placement in individual colonies, the transposonco-selection strategy was implemented to isolate and expand individualcolonies for DNA sequencing. Gene conversion using template from BelgianBlue cattle was detected in 5 colonies out of 366 examined by PCR.Amplification with primers outside the Belgian Blue HDR template andsequencing confirmed the presence of the expected 11 bp deletion in 4 ofthe colonies (FIG. 14). A second repeat experiment was performed withconsistent results, with about 1% of all tested colonies being positivefor bi-allelic conversion and about 0.5% to about 1% of all testedcolonies being heterozygous for allele conversion.

Example 7: TALEN Mediated DNA Cleavage as a Target for HDR in LivestockCells

TALEN pair LDLR2.1 (SEQ ID NO: 438 and 439) targeted to the fourth exonof the swine low density lipoprotein receptor (LDLR) gene (SEQ ID NO:349) was co-transfected with the supercoiled plasmid Ldlr-E4N-stop(Table 12, SEQ ID NO: 350) (designed to insert a stop codon into exon4), which contains homology arms corresponding to the swine LDLR geneand a gene-trap enabling expression of Neomycin phosphotransferase uponHDR (FIG. 15). After 3 days of culture PCR analysis revealed that, evenwithout antibiotic selection, a band corresponding to an HDR event canbe detected at the targeted locus (lane 4) at 30° C. Selection ofpopulations of cultured cells for 14 days with geneticin (G418) resultedin significant enrichment of HDR cells (lanes 11 & 13). Cloning of suchmodified cells by somatic cell nuclear transfer was performed, withsurrogate sows presently gestating the embryos.

TABLE 12 Description of sequence of the LDLR-E4N stop plasmid Name TypeMini . . . Maximum Len . . . 5′ Homology Arm misc_feat . . . 5,523 257899 left ITR LTR 5,374 5,514 141 pUC origin misc_feat . . . 4,701 5,368668 Amp resistance CDS 3,693 4,550 858 f1 ori misc_feat . . . 2,8683,174 307 Right ITR LTR 2,636 2,776 141 3′ Homology Arm misc_feat . . .2,330 2,627 298 Lox P misc_bindi . . . 2,283 2,316 34 Neo CDS 1,1861,977 792 ECMV IRES misc_feat . . . 576 1,185 610 Exon C_region 504 56663 BPSA splicing si . . . 401 506 106 Lox P misc_bindi . . . 330 363 34

Example 8: TALEN Activity in Bovine Zygotes

This Example compares results obtained with the Carlson +63 TALENS to a+231 scaffold. The methods described in Example 1 were followed. Table 2summarizes the results using the GDF83.1 TALEN pair (SEQ ID NOS: 428 and431) targeted to exon 3 of the bovine GDF8 locus, with the GDF83.1 beingbased on the Carlson +63 scaffold. Mutation frequency using the CARLSON+63 TALENs significantly exceeded previous injections. Six of 14blastocysts (43%) injected with a low mRNA dosage (2 ng/μ1) displayedindels without a significant reduction in development rate. Three offour blastocysts in the high dosage group (10 ng/μ1) displayed indels,with bi-allelic modification occurring in 2 of 3 mutant blastocysts(Table 3).

TABLE 3 Indels for GDF83.1 Bi-allelic Modification SEQ ID NOS SEQUENCES147 Wt. ACTCCACAGAATCTCGATGCTGTCGTTACCCTCTAACTGTGGATT 148 1a.ACTCCACAGAATCTCGATGCT:::GTTACCCTCTAACTGTGGATT X9 135 1b.ACTCCACAGAATCTCGATG:::::::::::CTCTAACTGTGGATT X2 136 1c.ACTCCACAGAATCTCGATGC::::::TACCCTCTAACTGTGGATT X1 137 2a.ACTCCACAGAAT:::::::::::::::::CCTCTAACTGTGGATT X3 138 2b.ACTCCACAGAATCTCGATGCT:::GTTACCTCTAACTGTGGATT X1 139 2c.ACTCCACAGAATCTCGATGCT:TCGTTACCCTCTAACTGTGGATT X2 2/9 Bi-allelic

Example 9: Cloning of TALEN-Modified Cells

Cells from Example 4 that were modified with LDLR2 TALEN pairs (SEQ IDNOS:438 and 439) were grown as clones. Transposon co-selected Ossabawswine colonies with mono- and bi-allelic modification of the Class Adomain 1 of the LDLR gene were pooled disproportionately (pools A—4genotypes, B—3 genotypes and C—5 genotypes) and cloned by chromatintransfer. Pregnancy was established in 7/9 transfers (½ for pool A, ⅔for pool B, and 4/4 for pool C). Seven of the 9 sows became pregnant,and 6 of the 7 pregnant sows had live births. 17 piglets were born thatappear to be in good health for purposes of raising to maturity. Thepiglets had various genotypes, referred to as B1, B2, C1 and C2 in Table4, below. Two of the genotypes were deletions, one was a single baseinsertion and one genotype had modifications of both alleles, aninsertion in one allele and deletion in the other.

TABLE 4 SEQ ID NOS. 141-145 SEQ ID NO SEQUENCE 140 Wt:CTCCTACAAGTGGATTGTGATGGGAACACCGAGTGCAAGGACGGGTCCG B1: (289_290INS34; 285_287delATG) 10 born; 9 live 141  1.CTCCTACAAGTGGATTTGTGATGGGA i34 ACACCGAGTGCAAGGACGGGTCCG 142  2.CTCCTACAAGTGGATTTGTG:::GGAACACCGAGTGCAAGGACGGGTCCG B2:(211_292del128) One stillborn 143  1.AGGGAGTATGGTCAC:::::::Δ128::ACCGAGTGCAAGGACGGGTCCG C1:(289_290del10) 3 born (one stillborn, one euthanized dueto clone defects) 144  1.CTCCTACAAGTGGATTTGTGATGGG::::::::::GCAAGGACGGGTCCG C2:(289_290insA) 8 born; 8 live 145  1. CTCCTACAAGTGGATTTGTGATGGG AAACACCGAGTGCAAGGACGGGTCCG

Example 10: An Adeno-Associated Virus (AAV) is an Effective Template forTALEN Stimulated Homologous Recombination (HR)

In another study, similar to Example 6, a mutant myostatin allele (11 bpdeletion) from Belgian Blue cattle was introgressed into the genome ofwild-type Wagyu cattle (Grobet, 1997, Kambadur, 1997) (FIG. 18). Fourmicrograms of TALEN encoding plasmids (SEQ ID NOS: 428 and 431) weretransfected into Wagyu cells and 24 hours later an adeno-associatedvirus (AAV-BB-HDR) harboring a 1,623 bp DNA fragment from Belgian Bluecattle was added. (SEQ ID NO: 505) Semi-quantitative PCR at day threesuggests an allele conversion frequency of up to 5% only when both GDF8TALENs and the AAV vector were added. To assess the frequency ofintrogression in individual colonies, the transposon co-selectionstrategy was implemented to isolate and expand individual colonies forDNA sequencing. Thirteen percent of isolated colonies were PCR positivefor introgression of the BB allele. These results demonstrate thatTALENs and an AAV homologous recombination template is an effectivemethod for targeted allele introgression in livestock and represents asignificant improvement over supercoiled plasmid homologousrecombination template for the same locus (Example 6).

Example 11: Single Stranded DNA for Templating

FIG. 19 summarizes the results of the transfection of TALEN encodingplasmids, containing an 11 base pair Belgian Blue cattle mutation, intoWagyu cells. Single stranded oligodeoxynucleotides (ssODNs) were foundto be an effective template for TALEN stimulated HR. The same loci asabove (Examples 6 and 10) were targeted to introgress the 11 base pairBelgian Blue cattle mutation into Wagyu cells. Two 76 base pair ssODNs(SEQ ID NOS: 501 and 502) were designed to mimic either the sense orantisense strand of the BB GDF8 gene including the 11 base pairdeletion. Four micrograms of TALEN encoding plasmids were transfectedinto Wagyu cells, and 0.3 nMol of ssODNs were either co-transfected withTALENS (N) or delivered 24 hours after TALEN nucleofection by eitherMirusLT1 (M) reagent or Lipofectamine LTX reagent (L). Semi-quantitativePCR at day three suggests an allele conversion frequency of up to 5% inconditions where ssODNs were delivered with LIPOFECTAMINE LTX reagent 24hours after TALEN transfection. No difference in PCR signal was observedbetween sense and antisense ssODNs designed against the target.

Example 12: Transfection of Livestock Cells with mRNAs Encoding TALENsResults in Efficient Target Cleavage

FIG. 20 summarizes the efficient target cleavage produced by thetransfection of TALEN encoding mRNAs into livestock cells. The TALENsequences can be found in Table 10. TALEN cDNAs (TALEN pairs p6511.1(SEQ ID NOS: 440, 441) and DMD7.1 (SEQ ID NOS: 410, 411)) were cloneddownstream of the T3 promoter in the pT3TS cloning vector transcribed asdescribed in Carlson, 2010 and purified using the MINELUTE PCRpurification kit (Qiagen) prior to mRNA synthesis using the MMESSAGEMACHINE T3 kit (Applied Biosciences) according to the manufacturersprotocol. Modified mRNA was synthesized from the same vectors with theMMESSAGE MACHINE T3 kit (Applied Biosciences) substituting aribonucleotide cocktail consisting of 3′-O-Me-m7G(5′)ppp(5′)G RNA capanalog (New England Biolabs), 5-methylcytidine triphosphatepseudouridine triphosphate (TriLink Biotechnologies, San Diego, Calif.)and two standard ribonucleotides, adenosine triphosphate and guanosinetriphosphate. mRNA synthesis reactions were DNAse treated prior topurification using the MEGACLEAR REACTION CLEANUP kit (AppliedBiosciences). a) The indicated quantities of p6511.1 TALENs weretransfected into pig fibroblasts (500,000-750,000 cells per replicate)using the NEON nucleofection system (Life Technologies) with thefollowing settings: 1 pulse, 1800 v; 20 ms width and a 100 μl tip.Transfected cells were culture 3 days at either 30 or 37 degrees Celsiusprior to indel analysis by the SURVEYOR assay (Transgenomic). PercentNHEJ was calculated as described in Guischin et. al., 2010, and plottedon the graph. Four micrograms of plasmid DNA (pDNA) encoding the p6511.1TALENs was also transfected under the same conditions for comparison of% NHEJ. b) mRNA structure, composition or in vitro synthesis reactionscheme have little effect on TALEN activity. mRNA encoding the DMD7.1TALENs was synthesized either by individually (“I” left and right TALENsin a separate reaction) or in the same reaction (Dual “D”) usingstandard or modified ribonucleotides. The reactions were then split intotwo replicates, one of which an additional polyA tail was added usingthe Poly(A) Tailing Kit (Ambion) according to the manufacturersprotocol.

Expression of TALENs from plasmid DNA has been an effective method forinduction of TALEN mediated indels in livestock cells; however,integration of the TALEN encoding plasmids into the genomes of cells ispossible. In contrast, mRNA cannot integrate into the genomes of hostcells. To avoid the integration of TALEN encoding plasmids, anexperiment was performed to determine if similar levels of TALENactivity could be achieved by transfection of mRNAs encoding TALENs.mRNA for TALENs encoding the p6511.1 TALEN pair was generated usingeither standard or modified ribonucleotides. Two quantities of eachTALEN mRNA preparation were transfected into pig fibroblasts bynucleofection, cultured 3 days at 30 or 37 degrees Celsius prior toanalysis of indels. Percent NHEJ was similar for all mRNA transfectionsincubated at 30 degrees Celsius while a dosage response could beobserved for transfected cells incubated at 37 degrees Celsius. Asignificant difference in percent NHEJ between modified and standardribonucleotides could not be detected in this replicate, however,equivalent quantities were not used. Notably, mRNA transfection in allgroups incubated at 30 degrees C. significantly outperformed the p6511.1TALENs transfected as plasmid DNA under the same conditions.

Another experiment was performed to examine the influence of modifiedversus standard nucleotide synthesized mRNA at a second locus, porcineDMD. This experiment also evaluated whether addition of a polyA tailinfluenced TALEN activity, and whether each TALEN monomer (left andright monomers) could be synthesized in the same transcription reaction(Dual) or if they must be synthesized individually and mixed prior totransfection. One or four micrograms of DMD7.1 TALEN mRNA weretransfected into pig fibroblasts and cultured 3 days at 30 or 37 degreesCelsius. As with the p6511.1 TALENs, little difference was observed inTALEN activity in cells cultured at 30 degrees Celsius suggesting thatneither modified nucleotides, in vitro poly adenylation of mRNAs or dualtranscription of mRNAs had an influence on activity. A dosage responsecould again be observed in the 37 degree cultured replicates as 4 μg ofmRNA outperformed 1 μg transfections. Also, polyadenylated mRNAsappeared to outperform non adenlyated mRNAs in 37 degree replicates.

Notably when plasmid DNA encoding the DMD7.1 TALENs was transfected intopig fibroblasts, a significant reduction (40-60%) in % NHEJ levelsmeasured at day 3 versus cells cultured to day 14 was noticed (Example3). No such reduction in % NHEJ was observed for any of the mRNAtransfected replicates shown here, data not shown for day 14modification levels. Thus mRNA transfection appears to be superior toDNA transfection not only for TALEN activity, but also for maintaining ahigh proportion of modified cells after an extended period in culture.Without being bound to a particular theory, it is believed that thisresult is due to improved cell viability when transfected with mRNAversus plasmid DNA.

Example 13: Analysis of Colonies Created by mRNA Transfection with NoSelection

One to four micrograms of mRNA encoding TALENs were added, as in Example12, to bovine or swine primary fibroblasts. The cells were grown at 30°C. for three days after exposure to TALENs and cells were enumerated andplated at a range of densities 1-20 cells/cm2 on 10 cm dishes. Cellswere cultured for 10-15 days until individual colonies of 3-4 mm indiameter could be observed. Colonies were aspirated with a p-200pipettor under gentle aspiration and expelled into a well of 24-wellplate with 500 μl of growth medium (Carlson, 2011). Plates with clearlydefined colonies (˜10-30/plate) were chosen for colony aspiration tolimit the chance of aspirating cells from multiple colonies. Once acolony reached 70-90 percent confluent in the 24-well dish, a portionwas harvested for indel analysis and the remainder was cryopreserved.The results of the indel analysis are located in the last five lines ofTable 5. These results demonstrate that colonies can be readily isolatedfrom TALEN mRNA transfected fibroblasts without the use of selectionmarkers. Mutation frequency in analyzed clones were accurately predictedby the modification levels of the source population at day 3. Cloneswith bi-allelic modifications could also be readily identified. Theresults of this Example are summarized in Table 5. The target sequencesare LDLR (SEQ ID NO: 242) (TALEN SEQ ID NOS: 438 and 439, 414-417), DMDexon 6 (SEQ ID NO:379) (TALEN SEQ NOS:434 and 437), DMD exon 7 (SEQ IDNO: 380) (TALEN SEQ ID NOS: 408-411), GHRHR (Gene ID 2692) (TALEN SEQ IDNOS:478 and 479), ACAN12 (SEQ ID NO: 378) (TALEN SEQ ID NOS: 401-404),and GDF8 (SEQ ID NOS: 351-353) (TALEN SEQ ID NOS:428 and 431).

TABLE 5 Genotype Distribution in Fibroblast Clones Table of Genotypedistribution in fibroblast clones. Observed Predicted % Predicted % ModClones Observed Bi- TALEN pair Selection Day 3 Mod Mod Clones Bi-allelicMod (%) allelic Mod (%) LDLRE2.1 Puro Pig ♂ 19 34.5 10.5 30/81 (37) 5/26 (19) LDLRE2.1 Puro Pig ♀ 21.5 38.3 12 23/76 (30)  8/23 (35)†LDLRE2.1 Puro Pig ♂ 14.4 26.7 7.7 12/94 (13)  2/12 (≥17)^(A)LDLRE2.1-2x^(B) Puro Pig 19.7 35.5 10.9  8/24 (33)  2/8 (≥25)^(A)LDLRE4.2 Puro Pig ♂ 20 36 11.1  4/48 (8.3)  ½ (25)^(A) LDLRE4.2 Puro Pig♀ 19 34.4 10  8/47 (17) 0/8^(A) DMDE6 Puro Pig 25 43.8 15.6 17/35 (49)NA DMDE7.1 Puro Pig 27 47 15.6 12/29 (41)  3/10 (30) DMDE7.1-2x^(B) PuroPig 22 39.2 12.4 22/41 (54)  7/22 (≥32)^(A)† GHRHR2.3 G-418 Pig 29 50 1726/43 (60) 15/26 (≥58)^(C)† ACAN12 Puro Cow 29 50 17 27/35 (77)  2/6(NA)^(D) btGDF83.1 Puro Cow 17 31 9.3  7/24 (29) 0/7 GHRHR2.3 None Pig ♂32.5 55 19.4 21/25 (84)  6/21 (≥29)^(A) GHRHR2.3 None Pig ♀ 35 58 21 13/13 (100)  3/13 (≥23)^(A) LDLR2.1 None Pig ♀ 34 57 20 88/166 (53)  5/16 (31%) btGDF83.1 None Cow 29 50 17 23/45 (51)  2/23 (≥9)^(E)btGDF83.1 None Cow 35 58 21 23/41 (56)  7/23 (≥30)^(E) ^(A)Bi-allelic KOwere identified by sequencing of PCR products. Only overlapping orhomozygous deletions can be identified using this technique.^(B)Fibroblasts were transfected and recovered twice within two weekswith the same TALEN pair. C 5/15 Bi-allelic colonies were confirmed asdouble frame-shift alleles. ^(D)Only colonies with distinguishable grossdeletions in the PCR amplicon were analyzed. ^(E)Bi-allelic KO colonieswere identified by high definition melt analysis. Only homozygousmodifications can be identified. †95% Confidence interval exceedsexpected bi-allelic null hypothesis

Example 14: Co-Transfection of mRNA Encoded TALENs and ssODNs EnhancesHDR

FIG. 21 sets forth a summary of experimental results for modifying Wagyucells with a combination of mRNA encoding TALENs and single-strandedoligonucleotides. The cells were Waygu cells and the allele was theBelgian Blue. Experimentation, cell type and locus assayed was the sameas in Example 11 with the exception that TALEN encoding mRNA (2 ug) wasdelivered in place of DNA encoded TALENs (where indicated) and only thesense ssODN was used. No selection markers were introduced in thesecells at any stage. Population analysis at day three revealed HDR inboth DNA and mRNA transfected cells when the ssODN was introduced 24hours after TALENs. Peak activity (˜10%) was observed in cellsco-transfected with the ssODN and TALEN mRNA by nuclofection. Thisresult contrasts the previous result with DNA encoding TALENs which wereunable to stimulate HDR at a measurable frequency. Among individualcolonies, prepared as in Example 12, both heterozygous and homozygousintrogression of the Belgian Blue allele could be observed at 5 and 2percent, respectively.

Example 15: Introduction of Single Base Alterations Using ssODNs andmRNA Encoding TALENs

FIGS. 22 and 23 depict results of another study wherein a singlenucleotide polymorphism was replicated. This polymorphism was known tocause a coding change to the bovine GDF8 locus, C313Y, known to causehypermuscularity in Piedmontese cattle (Kambadur, 1997; Genome Res. 19977: 910-915 An additional SNP was introduced into the ssODN to create asilent EcoRI site to aid in screening and/or quantification of HDR. mRNAand ssODNs were introduced simultaneously by nucleofection as indicatedin Examples 11 and 13 with relative quantities indicated. (SEQ ID NOS:503 and 431) FIGS. 22 and 23 show results when transfected cells areincubated at 30 and 37 degrees Celsius for three days. Peak levels ofhomologous recombination were significantly higher at 30 degrees Celsius(11.3%) than at 37 (1.7%), despite similar activity of the TALENs. (SEQID NOS: 442 and 443) The data shows that from about 0.2 to about 0.4nmol ssDNA is effective for homologous recombination both at 30 and 37degrees Celsius. Surprisingly, a bi-phasic effect was observed, with toogreat of an oligonucleotide concentration/amount abolishing therecombination. No selection markers were used in these cells at anystage.

Example 16: Alleles Introduced into Pig (Ossabaw) Cells Using Oligo HDR

FIG. 24 sets forth a summary of experimental results for modifying cellswith a combination of mRNA encoded TALENs and single-strandedoligonucleotides to place an allele that occurs naturally in one speciesto another species (interspecific migration). Piedmontese GFD8 SNP C313Y(SEQ ID NOS: 444 and 445), as in Example 15, was chosen as an exampleand was introduced into Ossabow swine cells. Experiments were performedas in Example 15 with Ossabaw swine cells substituted for Wagyu cells.No markers were used in these cells at any stage. A similar peak in HDRwas observed between pig and cattle cells at 0.4 nmol ssODN, (FIG. 22,23) however, HDR was not extinguished by higher concentrations of ssODNin Ossabaw fibroblasts.

Example 17: Cloning for Alleles Introduced into Cells Using Oligo HDR

FIG. 25 sets forth a summary of experimental results for modifying cellswith a combination of mRNA encoded TALENs and single-strandedoligonucleotides to place an allele into cells for cloning animals. Anew allele (BamH1) was introduced into Ossabaw swine cells designed tointroduce a 4 base pair insertion that would both create frame-shiftallele and introduce a novel BamH1 site. Two or 1 micrograms ofssLDLR2.1 TALEN mRNA (SEQ ID NOS: 438 and 439) and 0.3 nmol of ssODN wasintroduced into Ossabaw cells as in Examples 11 and 13. (SEQ ID NO: 506)Surprisingly, there was synergy between the ssODNs and the TALENactivity as the previous maximum for this TALEN pair without ssODN was25% NHEJ. This synergy was unexpected and not predictable based oncurrent understanding of the relevant molecular mechanisms. HDR wasdetected by restriction digest of PCR amplicons with BamH1. HDR levelswere similar to NHEJ levels suggesting that the majority of TALENinduced breaks were repaired with the ssODN. Analysis of individualcolonies, generated, as in example 13, revealed heterozygous andhomozygous modification of up to 30 and 2.5 percent respectively. Noselection markers were used in these animals at any stage. TALEN treatedcells were cloned by chromatin transfer, implanted into surrogate sowsand resulted in the establishment of pregnancies.

Example 18: DNA and mRNA Encoded TALENs are Active in SpermatigonialStem Cells

Results are summarized in FIG. 26. Porcine germ cells were isolated from10 wk old boars, and enriched by differential. Plasmids encoding eGFPand DMD-specific TALENs were transfected into germ cells using the AMAXANUCLEOFECTOR system Amaxa solutions “V”- and “L” and “B” using programsX-001 and X-005. (SEQ ID NOS: 504, 505, 506) Each transfection reactionwas performed with 10⁶ of enriched germ cells, and indicated microgramsof TALEN encoding plasmid DNA. The same methods were used to delivermRNAs encoding DMD7.1 TALENs. After nucleofection, the cells werecultured for 5 days in 5% CO₂ atmosphere at 37° C. or 30° C.Transfection efficiency was evaluated by immunofluorescence analysis forco-localization of expression of GFP and UCH-L1. Cell viability wasevaluated by trypan blue exclusion.

Example 19: TALEN Stimulated HDR in Primordial Germ Cells

TALEN stimulated HDR was also tested in chicken primordial germ cells(PGCs) at the chicken Ddx4 locus. Two TALEN pairs were constructed, onto intron 1 (Tal1.1) (SEQ ID NOS: 446 and 447) and exon 7 (Tal7.1) (SEQID NOS: 448 and 449) and their function was verified in DF1 chickencells, see FIG. 27. Tal1.1 lies within the homologous sequence of thedonor targeting vector. Tal7.1 lies outside the homologous sequence ofthe donor targeting vector. See also Example 7. Subsequently, each TALENpair was co-transfected with the donor targeting vector designed to fuseGFP with Exon 2 of the Ddx4 gene (Panel b). As expected cleavage withTal 1.1 stimulated homologous recombination (panel c) whereas Tal 7.,which lies outside of the homologous sequence in the donor targetingvector, did not stimulate HDR.

Example 20: TALEN Designing and Production

Candidate TALEN target DNA sequences and RVD sequences for examples20-40 were identified using the online tool “TAL EFFECTOR NUCLEOTIDETARGETER”. Plasmids for TALEN DNA transfection or in vitro TALEN mRNAtranscription were then constructed by following the Golden GateAssembly protocol using pCGOLDYTALEN (Addgene ID 38143) andRCIscript-GOLDYTALEN (Addgene ID 38143) as final destination vectors(Carlson 2012). The final pC-GoldyTALEN vectors were prepared by usingPureLink® HIPURE PLASMID MIDIPREP Kit (Life Technologies) and sequencedbefore usage. Assembled RCIscript vectors prepared using the QIAPREPSPIN MINIPREP kit (Qiagen) were linearized by SacI to be used astemplates for in vitro TALEN mRNA transcription using the mMESSAGEmMACHINE® T3 Kit (Ambion) as indicated elsewhere. Modified mRNA wassynthesized from RCIScript-GOLDYTALEN vectors as described in Carlson2012 substituting a ribonucleotide cocktail consisting of3′-0-Mem7G(5′)ppp(5′)G RNA cap analog (New England Biolabs),5-methylcytidine triphosphate pseudouridine triphosphate (TriLinkBiotechnologies, San Diego, Calif.) and adenosine triphosphate guanosinetriphosphate. Final nucleotide reaction concentrations are 6 mM for thecap analog, 1.5 mM for guanosine triphosphate, and 7.5 mM for the othernucleotides. Resulting mRNA was DNAse treated prior to purificationusing the MEGACLEAR REACTION CLEANUP kit (Applied Biosciences).

Example 21: CRISPR/Cas9 Design and Production

Gene specific gRNA sequences were cloned into the Church lab gRNA vector(Addgene ID: 41824) according their methods. The Cas9 nuclease wasprovided either by co-transfection of the hCas9 plasmid (Addgene ID:41815) or mRNA synthesized from RCIScript-hCas9. This RCIScript-hCas9was constructed by sub-cloning the XbaI-AgeI fragment from the hCas9plasmid (encompassing the hCas9 cDNA) into the RCIScript plasmid.Synthesis of mRNA was conducted as above except that linearization wasperformed using KpnI.

Example 22: Donor Repair Template Preparation

A) Bb-Hdr (1,623 Bp) Plasmid.

A 1,695 bp fragment encompassing the Belgian Blue allele was PCRamplified (btGDF8 BB 5-1: 5′-CAAAGTTGGTGACGTGACAGAGGTC (SEQ ID NO:328);btGDF8 BB 3-1: 5′-GTGTGCCATCCCTACTTTGTGGAA (SEQ ID NO:329)) from BelgianBlue genomic DNA and TOPO cloned into the PCR 2.1 vector (LifeTechnologies). This plasmid was used as positive control template foranalytical primer sets and for derivation of the 1,623 bp BB-HDRtemplate by PCR with following primers (BB del HR 1623 5-1:5′-GATGTATTCCTCAGACTTTTCC (SEQ ID NO:330); BB del HR 1623 3-1:5′-GTGGAATCTCATCTTACCAA, SEQ ID NO:331) and TOPO cloned as before. Eachplasmid was sequence verified prior to use. Transfection grade plasmidwas prepared using the Fast-Ion MIDI PLASMID ENDO-FREE kit (IBIScientific). rAAV packaging. BB-HDR was cloned into pAAV-MCS andpackaged into using the ADENO-ASSOCIATED VIRUS HELPER-FREE system(Agilent). Briefly, a 10 cm dish AAV-293 cells was transfected with 5 μgeach: pAAV-Helper, pAAV-RC and the AAV-BB-HDR plasmid. Two days posttransfection, the cells were removed from the plate by scraping into 1ml of growth media. Viral particles were released by 3 freeze-thawcycles prior to centrifugation at maximum speed in a microcentrifuge for5 minutes. The supernatant was aspirated and used directly for infectionof target cells.

B) Polled 1592 Template.

A 1,784 bp fragment encompassing 383 the POLLED allele was PCR amplified(F1: 5′-GGGCAAGTTGCTCAGCTGTTTTTG (SEQ ID NO:332);R1-5′-TCCGCATGGTTTAGCAGGATTCA, SEQ ID NO:333) from angus genomic DNA andTOPO cloned into the PCR 2.1 vector (Life Technologies). This plasmidwas used as positive the control template for analytical primer sets andfor derivation of the 1,592 bp HDR template by PCR with followingprimers (1594 F: 5′-ATCGAACCTGGGTCTTCTGCATTG SEQ ID NO:334; R1:5′-TCCGCATGGTTTAGCAGGATTCA, SEQ ID NO:335) and TOPO cloned as before.Each plasmid was sequence verified prior to use. Transfection gradeplasmid was prepared using the Fast-Ion MIDI Plasmid Endo-Free kit (IBIScientific) and 5 μg or 10 μg was transfected along with 2 μg HP1.3TALEN mRNA (SEQ ID NOS: 464 and 465) Oligonucleotide templates. Alloligonucleotide templates were synthesized by Integrated DNATechnologies, 100 nmole synthesis purified by standard desalting, andresuspended to 400 μM in TE.

Example 23: Tissue Culture and Transfection

Pig or cattle fibroblasts were maintained at 37 or 30° C. (as indicated)at 5% CO₂ in DMEM supplemented with 10% fetal bovine serum, 100 I.U./mlpenicillin and streptomycin, and 2 mM L-Glutamine. For transfection, allTALENs and HDR templates were delivered through transfection using theNeon Transfection system (Life Technologies) unless otherwise stated.Briefly, low passage Ossabaw, Landrace, Wagyu, or Holstein fibroblastsreaching 100% confluence were split 1:2 and harvested the next day at70-80% confluence. Each transfection was comprised of 500,000-600,000cells resuspended in buffer “R” mixed with plasmid DNA or mRNA andoligos and electroporated using the 100 μl tips by the followingparameters: input Voltage; 1800V; Pulse Width; 20 ms; and PulseNumber; 1. Typically, 2-4 μg of TALEN expression plasmid or 1-2 μg ofTALEN mRNA and 2-3 μM of oligos specific for the gene of interest wereincluded in each transfection. Deviation from those amounts is indicatedin the figure legends. After transfection, cells were divided 60:40 intotwo separate wells of a 6-well dish for three days' culture at either 30or 37° C. respectively. After three days, cell populations were expandedand at 37° C. until at least day 10 to assess stability of edits.

Example 24: Dilution Isolation of Cellular Clones

Three days post transfection, 50 to 250 cells were seeded onto 10 cmdishes and cultured until individual colonies reached about 5 mm indiameter. At this point, 6 ml of TrypLE (Life Technologies) 1:5(vol/vol) diluted in PBS was added and colonies were aspirated,transferred into wells of a 24-well dish well and cultured under thesame 420 conditions. Colonies reaching confluence were collected anddivided for cryopreservation and genotyping. Sample preparation:Transfected cells populations at day 3 and 10 were collected from a wellof a 6-well dish and 10-30% were resuspended in 50 μl of 1×PCRcompatible lysis buffer: 10 mM Tris-Cl pH 8.0, 2 mM EDTA, 0.45% TrytonX-100 (vol/vol), 0.45% Tween-20 (vol/vol) freshly supplemented with 200μg/ml Proteinase K. The lysates were processed in a thermal cycler usingthe following program: 55° C. for 60 minutes, 95° C. for 15 minutes.Colony samples from dilution cloning were treated as above using 20-30μl of lysis buffer.

Example 25: Plasmid and rAAV HDR in Wagyu Fibroblasts

Low passage Wagyu fibroblasts were cultured to 70-90% confluence andtransfected by NUCLEOFECTION (Lonza) with 2 μg each TALEN expressionplasmid (btGDF83.1L+NR, SEQ ID NO: 428) along with 750 ng of SLEEPINGBEAUTY transposon components as described in Carlson 2012. Forconditions where plasmid HDR template was used, 2 μg of BB-HDR plasmidwas also included in the transfection. Transfected cells were splitbetween two wells of a 6-well plate for culture at 30 or 37° C. Forconditions using rAAV HDR template, 150 μl of viral lysate was added toeach well 2 hours post transfection. After incubation for three days,cells were harvested by trypsinization, a portion of which were lysedfor analysis of HDR at day 3, and the remainder were plated for colonyisolation as described in Carlson 2012.

Example 26: Mutation Detection and RFLP Analysis

PCR flanking the intended sites was conducted using PLATINUM TAQ DNAPOLYMERASE HIFI (Life Technologies) with 1 μl of the cell lysateaccording to the manufacturer's recommendations. The frequency ofmutation in a population was analysed with the SURVEYOR MUTATIONDETECTION Kit (Transgenomic) according to the manufacturer'srecommendations using 10 ul of the PCR product as described above. RFLPanalysis was performed on 10 μl of the above PCR reaction using theindicated restriction enzyme. SURVEYOR and RFLP reactions were resolvedon a 10% TBE polyacrylamide gels and visualized by ethidium bromidestaining. Densitometry measurements of the bands were performed usingImageJ; and mutation rate of SURVEYOR reactions was calculated asdescribed in Guschin et al., 2010. Percent HDR was calculated viadividing the sum intensity of RFLP fragments by the sum intensity of theparental band+RFLP fragments. For analysis of mloxP insertion, small PCRproducts spanning the insertion site were resolved on 10% polyacrylamidegels and the insert versus wild type alleles could be distinguished bysize and quantified. RFLP analysis of colonies was treated similarlyexcept that the PCR products were amplified by 1×MYTAQ RED Mix (Bioline)and resolved on 2.5% agarose gels. For analysis of clones forintrogression of the GDF8 G938A-only (oligos lacked a novel RFLP),colonies were initially screened by a three primer assay that coulddistinguish between heterozygous ad homozygous introgression. Briefly,lysates from pig or cattle colonies were analysed by PCR using 1×MYTAQRED MIX (Bioline) using the following primers and programs. Cattle GDF8(Outside F1: 5′-CCTTGAGGTAGGAGAGTGTTTTGGG, SEQ ID NO:336, Outside R1:5′-TTCACCAGAAGACAAGGAGAATTGC, SEQ ID NO:337, Inside F1:5′-TAAGGCCAATTACTGCTCTGGAGACTA, SEQ ID NO:338; and 35 cycles of (95° C.,20 s; 62° C., 20 s; 72° C., 60s). Pig GDF8: Outside F1:5′-CCTTTTTAGAAGTCAAGGTAACAGACAC, SEQ ID NO:339, Outside R1:5′-TTGATTGGAGACATCTTTGTGGGAG, SEQ ID NO:340 Inside F1:5′-TAAGGCCAATTACTGCTCTGGAGATTA, SEQ ID NO:341; and 35 cycles of (95° C.,20 s; 58° C., 20 s; 72° C., 60s). Amplicons from candidates weresequenced directly and/or TOPO cloned (Life Technologies) and sequencedby Sanger sequencing. To detect TALEN-mediated HDR at with the BB-HDRtemplate, either 1 μl or 1 μl of a 1:10 dilution of PCR-lysate (1,000cells/ul) was added to a PCR reaction with PCR primers bt GDF8 BB 5-1(primer “c”) and primer “c” (BB-Detect 3-1-5′-GCATCGAGATTCTGTCACAATCAA,SEQ ID NO:342) and subjected to PCR with using 1× MYTAQ RED MIX(Bioline) for 40 cycles (9 459 5° C., 20 s; 66° C., 20 s; 72° C., 60s).To confirm HDR in colonies identified by the above PCR, amplification ofthe entire locus was performed with primers bt GDF8 BB 5-1 and bt GDF8BB 3-1 followed by TOPO cloning (Life Technologies) and sequencing.

Example 27: Detection of POLLED Introgression

Detection of POLLED introgression was performed by PCR using the F1primer (see above) and the “P” primer (5′-ACGTACTCTTCATTTCACAGCCTAC, SEQID NO:343) using 1× MyTaq Red mix (Bioline) for 38 cycles (95° C., 25s;62° C., 25s; 72° C., 60s). A second PCR assay was performed using (F2:5′-GTCTGGGGTGAGATAGTTTTCTTGG, SEQ ID NO:344;R2-5′-GGCAGAGATGTTGGTCTTGGGTGT, SEQ ID NO:345). Candidates passing bothtests were analysed by PCR using the flanking F1 and R1 primers followedby TOPO cloning and sequencing. Detection of FecB introgression wasperformed as previously described for sheep. Callipyge introgression wasdetected by an AVAII RFLP assay. See results in FIG. 35.

Example 28: Amplicon Sequencing and Analysis

DNA was isolated from transfected populations and 100-250 ng was addedto a 50 μl PLATINUM TAQ DNA POLYMERASE HIGH FIDELITY (Life Technologies)assembled per the manufacturer's recommendations. Each sample wasassigned a primer set with a unique barcode to enable multiplexsequencing. A portion of the PCR product was resolved on a 2.5% agarosegel to confirm size prior to PCR cleanup using the MINELUTE PCRPURIFICATION Kit (Qiagen). Samples were quantified and pooled into asingle sample for sequencing. The single combined sample was spiked with25% PhiX (for sequence diversity) and sequenced on an Illumina MISEQsequencer generating 150 base-pair paired-end reads. Read quality wasassessed using FASTQC Read-pairs with overlapping ends were joined usingFASTQ-JOIN from the EA-UTILS package. A custom PERL script was used todemultiplex the joined reads and count insert types. Exact matches tothe forward and reverse primers were required in the demultiplexingstep. Cloned animals were genotyped by RFLP assay and sequencing.

Example 29 Evaluation of Transfected mRNA as a Source of TALENs

Referring to FIG. 36, TALENs were introduced into pig fibroblastsencoded by either unmodified mRNA, modified mRNA (mod mRNA) or plasmidDNA (pDNA). Two quantities of each TALEN preparation were transfectedinto cells by nucleofection (Lonza), cultured 3 days at 30° C. or 37° C.prior to analysis of indels. Percent NHEJ was similar for all mRNAtransfections incubated at 30° C., while a dosage response could beobserved for transfected cells incubated at 37° C. Notably, mRNAtransfection in all groups incubated at 30° C. significantlyoutperformed the TALENs transfected as plasmid DNA under the sameconditions. There was little difference between modified and unmodifiedmRNA in this test. TALENs:CTCCTCCATTGCGGACATGGACTTCTCAGCCCTTCTGAGTCAGATC (SEQ ID NO:346),underlines indicate TALENs binding sites.

Example 30: Kinetics of TALEN Induced HDR with Oligonucleotide Templates

Referring to FIG. 37A, an mRNA source of TALENs stimulated efficient andconsistent HDR using an oligo donor. Each chart displays results oftargeting a specific locus in fibroblasts (e.g., ssIL2RG; “ss” for Susscrofa and “bt” for Bos taurus) using oligo donor templates and TALENsdelivered as plasmid DNA or mRNA. (Insets) ssIL2RG2.1 (SEQ. ID NOS: 484and 485); ssRAG2.1 (SEQ ID NOS: 488 and 489); btGGTA9.1 (SEQ ID NOS: 490and 491); ssLDLR2.1 (SEQ ID NOS: 438 and 439) Diagrams of the oligotemplates, in which the shaded boxes represent the TALEN-binding siteand the spacers are shown in white. Each oligo contains either a 4-bpinsertion (ins4) or deletion (del4) that introduces a novel restrictionsite for RFLP analysis. Presumptive BMs replace the conserved −1thymidine (relative to the TALEN-binding site) with the indicatednucleotide. Fibroblasts were transfected with either TALEN-encodingplasmids (3 μg) or mRNA (1 μg) along with 3 μM of their cognateoligo-homologous template. Cells were then incubated at 37° C. or 30° C.for 3 d before expansion at 37° C. until day 10. TALEN activity wasmeasured by the Surveyor assay at day 3 (Day3 Surveyor), and HDR wasmeasured at days 3 and 10 by RFLP analysis (Day3% HDR and Day10% HDR).Each bar displays the average and SEM from three replicates.

Referring to FIG. 37B, porcine fibroblasts were transfected with eitherTALEN-encoding mRNA or plasmid DNA and oligos with 4 base pairinsertions targeting LDLR or APC genes. Cells from each transfectionwere then evenly split into seven 24-well plate wells, cultured at 30°C. and assayed by RFLP at the indicated time points. Panel a) RFLPanalysis on cell populations at indicated time points. Panel b) Resultsfrom panel a were quantified by densitometry and the averages wereplotted as a function of time with SEM (n=3). HDR signal first appears12 hours post-transfection and accumulates over time. The onset of HDRat LDLR was independent of TALEN source, but the rate of HDR between 24and 72 hours was much higher when mRNA was used compared to plasmid DNA.

Example 31: Influence of Mutation Type on the Frequency of HDR

Referring to FIG. 38, panel a) The sequence of five oligos used totarget ssLDLR. Oligos vary in length and type of mutation. TALEN bindingsites are indicated in boxed text and the novel BamHI site isunderlined. SNPs including BMs and insertions are circled. Panel b)Cells were transfected with LDLR2.1 TALEN mRNA (1 μg) and oligos (2 μMfinal). HDR at day 3 was determined by RFLP analysis and the averagewith SEM (n=3) was plotted. The results suggest that insertion allelesare more efficiently incorporated than SNPs or deletions, but thathomology length from 46-90 bp has negligible influence on HDRefficiency. c) Cattle cells were transfected with btRosa1.2 TALEN mRNAand either 41_mloxP or 60_loxP oligos (2 μM final). The numbers 41 and60 refer to the number of homologous bases. Each oligo contains a 34 bploxP site, either a modified (mloxP) or wild type (loxP) version, in thecenter of the spacer. Densitometry at day 3 and 15 show that insertionof loxP sites is both efficient and stable.

Example 32: CRISPR/Cas9 Mediated HDR to Introgress the p65 S531PMutation from Warthogs into Conventional Swine

Referring to FIG. 39, panel a) The S531P missense mutation is caused bya T-C transition at nucleotide 1591 of porcine p65. The S-P HDR templateincludes the causative TC transition mutation (oversized text) whichintroduces a novel XmaI site and enables RFLP screening. Two gRNAsequences (P65_G1S and P65_G2A) are shown along with the p65.8 TALENsused in previous experiments. Panel b) Landrace fibroblasts weretransfected with S—P-HDR oligos (2 μM), two quantities of plasmidencoding hCas9 (0.5 μg or 2.0 m); and five quantities of the G2Atranscription plasmid (0.05 to 1.0 μg). Cells from each transfectionwere split 60:40 for culture at 30 and 37° C. respectively for 3 daysbefore prolonged culture at 37° C. until day 10. Surveyor assay revealedactivity ranging from 16-30%. Panels c and d) RFLP analysis of cellssampled at days 3 and 10. Expected cleavage products of 191 and 118 bpare indicated by black arrows. Despite close proximity of the doublestranded break (DSB) to the target SNP, CRISPR/Cas9 mediated HDR wasless efficient than TALENs for introgression of S531P. Individualcolonies were also analyzed using each gRNA sequence (data not shown).

Referring to FIG. 40, experiments were made for comparison of TALENs andCRISPR/Cas9 mediated HDR at porcine APC. Panel a) APC14.2 TALENs and thegRNA sequence APC14.2 G1a are shown relative to the wild type APCsequence. Below, the HDR oligo is shown which delivers a 4 bp insertion(orange text) resulting in a novel HindIII site. Pig fibroblaststransfected with 2 μM of oligo HDR template, and either 1 μg TALEN mRNA,1 μg each plasmid DNA encoding hCas9 and the gRNA expression plasmid; or1 μg mRNA encoding hCas9 and 0.5 μg of gRNA expression plasmid, werethen split and cultured at either 30 or 37° C. for 3 days beforeexpansion at 37° C. until day 10. Panel b) Charts displaying RFLP andSurveyor assay results. TALEN stimulated HDR was most efficient at 30°C., while CRISPR/Cas9 mediated HDR was most effective at 37° C. For thislocus, TALENs were more effective than the CRISPR/Cas9 system forstimulation of HDR despite similar DNA cutting frequency measured bySurveyor assay. In contrast to TALENs, there was little difference inHDR when hCas9 was delivered as mRNA versus plasmid.

Example 33: SNP Introgression Using Oligo Donors

Referring to FIG. 41, panel a) The influence of blocking mutations (BM)on maintenance of HDR alleles was evaluated in pig LDLR and GDF8. Eacholigo was designed to introduce the same SNPs/restriction 313 site plusor minus blocking mutations. HR was quantified in transfectedpopulations initially cultured at 30° C. for three days and furthermaintained at 37° C. until day 12 by RFLP assay. The average and SEM(n=3) is shown. Panel b) Introgression of myostatin C313Y into Wagyufibroblasts. The C313Y missense mutation is caused by a G-A transition(indicated by oversized text) at nucleotide 938 of bovine myostatin TheHDR template also includes a T to C transition (circled) to introduce anovel EcoRI site for RFLP screening. Two left TALENs were designedagainst the locus, btGDF83.6-G, targeting the wild type alelle (Wt), andbtGDF83.6-A targeting the mutant allele (C313Y); both share a commonright TALEN. Transfection, culture and measurement were conducted asabove. The average and SEM for btGDF83.6-G (n=30) and btGDF83.6-A (n=5)represent twelve and three biological replicates, respectively. Atwo-sided student's t-test was used to compare averages between groups;the p values are indicated.

Example 34: SNPs

FIG. 42 is a plot that shows results for sequence analysis of TALENstimulated HDR alleles. PCR amplicons flanking the target site (200-250bp total) derived from TALEN mRNA and oligo transfected cell populationswere sequenced by ILLUMINA sequencing. Total read count ranged from10,000 328 to 400,000 per sample. The count of perfect, intended HRreads versus the wild type reads is plotted for insertion (panel a) andSNP alleles (panel b). The target locus, time point and whether or notBMs were included in the oligo are indicated below. Panel c). Reads frombtGDF8 and p65 were sorted for incorporation of the target SNP and thenclassified intended (iSNP) versus those with an additional mutation(iSNP+Mut) and plotted against the total number of reads.

Example 35: Sequence Analysis of HDR Alleles

Referring to FIG. 43, sequencing reads containing the correct insertion(Panel a) or SNP allele (Panel b) were analyzed for incorporation of BM.The target locus, time point and whether or not BMs were included in theoligo are indicated below each graph. In general, the 5′ BM wasincorporated most frequently into the HDR conversion tract, followed byinclusion of both BMs, or the 3′ BM only. The distribution of BM issomewhat skewed towards incorporation of both BM when the intendedmutation to LDLR is a SNP versus a 4 bp insertion allele. It is alsointeresting to note that the majority of intended reads for btGDF8 haveincorporated at least one BM, but seldom have the 3′ BM alone. Thus,while BM did not have a significant impact on the frequency ofmaintaining the intended SNP (iSNP) allele in culture, their enrichmentrelative to other loci suggests that they might have offered someprotection from TALEN re-cleavage. c). The data of FIG. 42 panel c wasfurther classified by mutation type and compared. Some reads containedonly the iSNP, others had a concomitant indel (iSNP+indel), or aconcomitant unintended SNP (iSNP+uSNP). There appears to be someelevation in the frequency of iSNP+indel when BMs were not included inthe template, and the majority of indels were located in the spacerregion so are likely to be the result of re-cutting of already convertedalleles.

Example 36: Multiple SNPs in the TALEN DNA-Binding Site Stabilize HDRAlleles

Referring to FIG. 44, the EIF4GI gene was stabilized with multiple SNPsin the TALEN DNA binding site. Panel a) A portion of wild type EIF4GIWt-NL is shown. One pair of TALENs was designed to cut the wild typeEIF4GI to stimulate homologous recombination. Also aligned to the Wtsequence is the core sequence of the donor oligo, DF-HDR, used tointroduce three SNPs (red oversized letters) into the genome. The thirdSNP creates a novel EagI restriction site that was used for RFLPanalysis. Pig fibroblasts were transfected with EIF4GI14.1 TALEN mRNA (2μg) and DF-HDR (2 μM) and then cultured at 30° C. for 3 days prior toanalysis and colony propagation. Panel b) RFLP analysis on populationthree days post transfection. Expected product sizes of 344, 177 and 167bp are indicated by filled triangles. Panel c) RFLP assay on isolatedcellular clones. Day 3 cells were used to derive monoclonal coloniesthrough dilution cloning. An example of colonies with heterozygous (opentriangles) or homozygous (filled triangles) HDR alleles are indicated.

Example 37: Hypothermic Treatment for Maintenance of SNP HDR Alleles

Referring to FIG. 45, pig fibroblasts were transfected with TALEN mRNA(1 μg) and oligos (3 μM). Cells from two independent transfections werepooled for each replicate and evenly distributed into six wells of a6-well plate and cultured at 30° C. Samples were collected from thesepopulations for RFLP analysis on days 1-7 (minus day 6, 1D to 7D alongX-axis) post-transfection and the remaining cells were transferred to37° C. Samples for each condition were collected again at day 12 forRFLP analysis. The average HDR and SEM (n=3) is shown at the initialcollection and once again at day 12.

Example 38: Intentional RVD Mismatches for Introgression of SNPs

Referring to FIG. 46, panel a) A TALEN pair (caCLPG 1.1) was designed totarget the caCLPG region. Oligo driven HDR was utilized to introduce thedesired Adenine to Guanine SNP (the targeted Adenine is boxed). Thedesired SNP allowed genotyping by a loss of an AvaII restriction site.Each TALEN monomer is indicated in shading above their respectivebinding locations. The N- and C-termini are indicated with N and C,respectively. b) Each allele of single-cell derived colonies that wereresistant to AvaII were sequenced (only AvaII resistant alleles areshown). All of the alleles that contained our SNP or interest (boxed)also contained deletions (marked with dashes in the AvaII ResistantAllele sequences) or insertions (marked with dashes in the WT sequence).c) To reduce the possibility of re-binding, and subsequently re-cutting,intentional mismatches (italicized circled text) were introduced intothe RVD sequence. The mismatches were placed in the RVDs directly beforeand/or after the RVD that would bind to the desired SNP (boxed) in rightmonomer of the TALEN. d) TALEN activity was measured via a Cell assay.The percent of non-homologous end joining (% NHEJ) was equivalent for1.1 and 1.1b (28%), but was greater than 1.1 for 1.1a and 1.1c (30% and31% respectively). The no-RNA negative control showed no TALEN activity(0%). e) Both alleles of AvaII-resistant single-cell derived coloniesproduced with caCLPG 1.1c were sequenced. The desired SNP is boxed.Colony 37 and 78 were heterozygous for the desired SNP and showed noadditional indels. Colony 142 was homozygous for the desired SNP, butcontained a 4 bp insertion on one allele.

Example 39: Mismatch Required for SNP Introgression

Referring to FIG. 47, a mismatch was required for SNP introgression. Aschematic of the bovine DGAT sequence around K323A. The grey arrowsrepresent the TALEN monomers where they bind to the DGAT sequence. Theleft arm consists of 16 RVDs, the right arm consists of 15 RVDs, and thespacer is 16 base pairs long. The GC and ggagct, boxed, are the targetedbase pairs. The DGAT oligo converts the GC to an AA to create thedesired DGAT mutant. As a marker for HDR, the boxed GGGAGC is convertedto AAGCTT that creates a novel HindIII restriction site. Since thischange is in the spacer, it should not affect TALEN binding as to notinterfere with the intentional mismatch results. b) DGAT TALEN RVDsequences. btDGAT 14.2 contains no intentional mismatches in the RVDs.btDGAT 14.4, 14.5, and 14.6 each contain one intentional RVD mismatch ateither position 1, 3, or 5 of the left TALEN monomer (circled). c)Bovine fibroblasts were transfected with 1 ug of talen and 0.4 nmoles ofoligo. Three days after transfection cells were lysed, the DGAT sequencewas amplified by PCR, digested with HindIII and ran on an acrylamidegel. The percent efficiency of HDR was determined by densitometry (HR).d) Sequence analysis of colonies produce with the original 14.2 TALENs.Of twelve colonies, none that were positive for the HindIII RFLPcontained the desired mutation due to indels overlapping the site. e)Colonies derived from TALENs 14.5 and 14.6 produced the correct DGATmutation and HindIII restriction site. These two TALEN pairs produced atotal of two homozygous (HH) and three heterozygous (Hh) colonies. TALEN14.4 did not produce any colonies with the correct DGAT mutation (datanot shown).

Example 40: All-in-One TALEN-HDR/Cre-RMCE

FIG. 48 depicts a process of TALEN-HDR/RMCE. The foxed cassette istransfected along with TALENs compatible with the oligo, the loxP oligoand a source of Cre recombinase. For this process to work, TALENs mustcut the target loci followed by repair with the loxP oligo prior toCre-mediated RMCE into the repaired site. The bar graph shows the numberof puromycin resistant colonies produced by this method when YFC-Creversus mCherry was included in the transfection. To confirm targeting tothe SRY locus, PCR was conducted across the predicted junction (asindicated) will result in a 370 bp product. This product is apparentonly when Cre is included. For this set of experiments, the followingconditions were used: 600,000 cells transfected with 1 ug SRY TALENs+0.3nMol of SSCY_LoxP oligo+CLP-YFP-Cre (0.5 ug)+Floxed PTK (2 ug). Thenegative control had 0.5 ug of mCherry plasmid in place of CLP-YFP-Cre.SSCY_LoxP oligo:

(SEQ ID NO: 320) TTTTATATACATTTTACACACATATATATGAAACATAACTTCGTATAGGAGACTTTATACGAAGTTATGGATCCAAGCTTATAACTTCGTATAATGTATGCTATACGAAGTTATTGACAGTATTAATGGCCTGAACCTAGCCAGAACT

Example 41: Confirmation of Belgian Blue Introgression by Sequencing

The schematics of Wagyu wild-type GDF8 and the Belgian Blue template(BB-HDR) are shown in FIG. 50. PCR was conducted using primers locatedoutside of the homology arms (c and d) on five PCR positive coloniesfollowed by cloning and sequencing with primer b′. Comparison to thewild-type sequence reveals the expected 11-basepair deletioncharacteristic the Belgian Blue allele (heterozygous) in 4 of 5colonies. TALENs (btGDF83.1, SEQ ID NOS:428 and 431) and a dsDNAtemplate (BB-HDR) were designed to introduce an 11-basepair deletioninto exon-3 of bovine GDF8 (Belgium Blue mutation) by double-strandbreak-induced homologous recombination. (SEQ ID NO: 504) Half of thebinding site for the left TALEN is missing in the BB-HDR template andthus should be resistant to TALEN cleavage. SURVEYOR assay demonstratedactivity of btGDF83.1 TALENs at both 37 and 30° Celsius. Allele-specificPCR demonstrated that HDR induction was dependent on co-transfection ofTALENs and the BB-HDR template. The PCR assay was developed tospecifically detect HDR modified GDF8 alleles using primers c and c′.The 3′ end of primer c′ spans the 11-basepair deletion, and cannotamplify the wild type allele (wt). Five hundred cell equivalents wereincluded in each PCR reaction including the positive control. PercentHDR was determined by comparative densitometry between experimental andcontrol reactions.

Example 42: Precision Alteration of Intended Gene in Wild-Type WagyuCattle

A gene of wild-type Wagyu cattle was altered by making a deletion in atargeted area of the gene (an 11 bp deletion) in Wagyu fibroblasts ascan be seen in FIG. 50. This alteration made the Wagyu cattle have theallele of Beligan Blue cattle. When transfected alone, the btGDF8.1TALEN pair (SEQ ID NO: 428 and 431) cleaved up to 16% of chromosomes atthe target locus. TALENs (btGDF83.1) and a dsDNA template (BB-HDR) weredesigned to introduce an 11-bp deletion in exon-3 of bovine GDF8(Belgium Blue mutation) by DSB induced homologous recombination. (SEQ IDNO: 504) Half of the binding site for the left TALEN was missing in theBB-HDR template, to make it resistant to TALEN cleavage. A SURVEYORassay demonstrated activity of btGDF83.1 TALENs at both 37 and 30°Celsius. The PCR product used for this assay was generated using primersb and b′ (not shown). The BB-HDR template was not included in thesereplicates since it would confound estimates of btGDF83.1 activity.Allele specific PCR demonstrated that HDR induction was dependent onco-transfection of TALENs and the BB-HDR template. The PCR assay wasdeveloped to specifically detect HDR modified GDF8 alleles using primersc and c′ (not shown). The 3′ end of primer c′ spanned the 11-bp deletionso that it could not amplify the wild type allele “wt”. Five hundredcell equivalents were included in each PCR reaction including thepositive control “C”. Percent HDR was determined by comparativedensitometry between experimental and control reactions. Co-transfectionwith a supercoiled DNA template harboring a 1623 bp DNA fragment fromBelgian Blue cattle resulted in a gene conversion frequency (HDR) of0.5% to 5% as suggested by semi-quantitative PCR at day 3, withoutselection for the desired event. These results demonstrated that TALENscan be used to effectively place exogenous nucleic acid sequences inlivestock, including alleles—and without markers. To assess thefrequency of placement in individual colonies, the transposonco-selection strategy was implemented to isolate and expand individualcolonies for DNA sequencing. Gene conversion using template from BelgianBlue cattle was detected in 5 colonies out of 366 examined by PCR.Amplification with primers outside the Belgian Blue HDR template andsequencing confirmed the presence of the expected 11 bp deletion in 4 ofthe colonies.

A second repeat experiment was performed with consistent results, withabout 1% of all tested colonies being positive for bi-allelic conversionand about 0.5% to about 1% of all tested colonies being heterozygous forallele conversion.

Similarly, alleles were also introduced into pig (Ossabaw) cells usingoligo HDR. The cells were modified with a combination of mRNA encodedTALENs and single-stranded oligonucleotides to place an allele thatoccurs naturally in one species to another species (interspecificmigration) as can be seen in FIG. 51. Piedmontese GDF8 SNP C313Y, werechosen as an example and was introduced into Ossabow swine cells. Nomarkers were used in these cells at any stage. A similar peak in HDR wasobserved between pig and cattle cells at 0.4 nmol ssODN, (not shown)however, HDR was not extinguished by higher concentrations of ssODN inOssabaw fibroblasts. (SEQ ID NOS: 444, 445 and 507)

Example 43: Modification at Intended Targets

Consistent modification of targeted genes was made. Referring to FIG.52, each chart displays results of targeting a specific locus infibroblasts (e.g., ssIL2RG; “ss” for Sus scrofa and “bt” for Bos taurus)using oligo donor templates and TALENs delivered as plasmid DNA or mRNA.(Insets) Diagrams of the oligo templates, in which the shaded boxesrepresent the TALEN-binding site and the spacers are shown in white.Each oligo contains either a 4-bp insertion (ins4) or deletion (del4)that introduces a novel restriction site for RFLP analysis, see Table 15below. (SEQ ID NOS: 508-513) Presumptive blocking mutations (BM) replacethe conserved −1 thymidine (relative to the TALEN-binding site) with theindicated nucleotide. Fibroblasts were transfected with eitherTALEN-encoding plasmids (3 μg) or mRNA (1 μg) along with 3 μM of theircognate oligo-homologous template. Cells were then incubated at 37° C.or 30° C. for 3 d before expansion at 37° C. until day 10. TALENactivity was measured by the Surveyor assay at day 3 (Day3 Surveyor),and HDR was measured at days 3 and 10 by RFLP analysis (Day3% HDR andDay10% HDR). Each bar displays the average and SEM from threereplicates. Each of the targeted loci was successfully modified. TheTALEN pairs used were ssILRG2.1 (SEQ ID NOS: 484 and 485); ssRAG2.1 (SEQID NOS:488 and 489); btGGTA9.1 (SEQ ID NOS:490 and 491); and ssLDLR2.1(SEQ ID NOS:438 and 439).

TABLE 15 Oligonucleotide HDR templates TALEN FIG./ pair panelssODN design Sequence ssLDLR2.1 46_SNPsCCTACAAGTGGATTTGTGGGATCCACACCGAGTGCAA BamHI GGACGGGTC (SEQ ID NO: 508)ssLDLR2.1 53B 90_SNPs TGCCGAGACGGGAAATGCATCTCCTACAAGTGGATTT BamHIGTGGGATCCACACCGAGTGCAAGGACGGGTCCGATG AGTCCCTGGAGACGTGC (SEQ ID NO: 509)ssLDLR2.1 53A 90_ins4_BM CCGAGACGGGAAATGCACCTCCTACAAGTGGATTTGT BamHIGATGGATCCGAACACCGAGTGCAAGGACGGGTCCGC TGAGTCCCTGGAGACGT (SEQ ID NO: 510)ssLDLR2.1 53B 90_SNPs_BM TGCCGAGACGGGAAATGCACCTCCTACAAGTGGATTT BamH1GTGGGATCCACACCGAGTGCAAGGACGGGTCCGCTG AGTCCCTGGAGACGTGC (SEQ ID NO: 511)ssLDLR2.1 60_SNPs_BM TGCACCTCCTACAAGTGGATTTGTGGGATCCACACCG BamH1AGTGCAAGGACGGGTCCGCTGAG (SEQ ID NO: 512) ssLDLR2.1 86_del4_BMTGCCGAGACGGGAAATGCACCTCCTACAAGTGGATTT BamH1GGGATCCACCGAGTGCAAGGACGGGTCCGCTGAGTC CCTGGAGACGTGC (SEQ ID NO: 513)ssAPC14.2 53A 90_ins4_BM CCAGATCGCCAAAGTCACGGAAGAAGTATCAGCCAT HindIIITCATCCCTCCCAGTGAAGCTTACAGAAATTCTGGGTC GACCACGGAGTTGCACT (SEQ ID NO: 514)ssTp53 53A 90_ins5_BM AGCTCGCCACCCCCGCCGGGCACCCGTGTCCGCGCCA HindIIITGGCCATCTAAGCTTAAAGAAGTCAGAGTACATGCCC GAGGTGGTGAGGCGCT (SEQ ID NO: 515)btGDF83. 53B 90_SNPs_BM CTAAAAGATATAAGGCCAATTACCGCTCTGGAGAATA 6-G EcoRITGAATTCGTATTTTTGCAAAAGTATCCTCATCCCCATC TTGTGCACCAAGCAA (SEQ ID NO: 516)btGDF83. 53B 90_SNP CTAAAAGATATAAGGCCAATTACTGCTCTGGAGAATA 6-GTGAATTTGTATTTTTGCAAAAGTATCCTCATACCCATC TTGTGCACCAAGCAA (SEQ ID NO: 517)ssP65.8 53B 90_SNP GGGCCTCTGGGCTCACCAACGGTCTCCTCCCGGGGGA XmaICGAAGACTTCTCCTCCATTGCGGACATGGACTTCTCA GCCCTTCTGAGTCAGA (SEQ ID NO: 518)

Example 44: High Efficiency for Making Intended Changes in Genes

FIG. 53 shows analysis of changes made to genes APC (SEQ ID NOS:482 and483), LDLR (SEQ ID NOS:438 and 439), p53 (SEQ ID NOS:452 and 453), p65(SEQ ID NOS:440 and 441), and btGDF8 (SEQ ID NOS:428 and 431) (TALENpairs shown in parentheses). FIG. 53 shows three graphs: (a) shows datafrom pig fibroblasts, (b) shows data from pig fibroblasts (LDLR, p65)and cattle fibroblasts (DGF8), and (c) shows data from pig fibroblasts(p65) and cattle fibroblasts 9GDF8). In some cases insertions wereintended, while SNPs were intended in other cases. Changes were madewith TALENs and HDR templates, as described above. Table 15, above,lists the HDR templates. (SEQ ID NOS: 509-511 and 514-518) The count ofperfect, intended HR reads versus the wild type reads is plotted for:insertion (panel a) and SNP alleles (panel b). Sequence analysis ofTALEN stimulated HDR alleles was made. PCR amplicons flanking the targetsite (200-250 bp total) derived from TALEN mRNA and oligo transfectedcell populations were sequenced by ILLUMINA sequencing. Total read countranged from 10,000 to 400,000 per sample. The target locus, time pointand whether or not BMs were included in the oligo are indicated below.Panel c shows reads from btGDF8 and p65, as sorted for incorporation ofthe target SNP and then classified intended (iSNP) versus those with anadditional mutation (iSNP+Mut) and plotted against the total number ofreads. Accordingly, in the case where only a single SNP was intended,there were also additional changes, as indicated.

Example 45: Frequencies for Recovery of Colonies with HDR Alleles

Table 7, entitled Frequencies for recovery of colonies with HDR alleles,lists the results of an analysis of about 650 colonies of cells forintended indel alleles in eight separate loci. The analysis revealed arecovery rate of 10-64% (average, 45%), with up to 32% of the colonieshomozygous for the edit. Changes were made with TALENs and HDRtemplates, as described above. The colonies were obtained by dilutioncloning without drug selection.

Example 46: Cloned Pigs with HDR Alleles of DAZL and APC

FIG. 54 shows a genetic analysis of cloned animals. Two gene-edited lociin the porcine genome, deleted in azoospermia-like (DAZL, SEQ ID NOS:182-183) and adenomatous polyposis coli (APC, SEQ ID NOS:188-189) werechosen. Colonies of cultured cells treated with HDR- or NHEJ editedalleles of DAZL or APC were pooled for cloning by chromatin transfer(CT). Each pool yielded two pregnancies from three transfers, of whichone pregnancy each was carried to term. A total of eight piglets wereborn from DAZL-modified cells, white composite pig fibroblasts, each ofwhich reflected genotypes of the chosen colonies consistent with eitherthe HDR allele (founders 1650, 1651, and 1657) or deletions resultingfrom NHEJ (FIG. 53A, FIG. 54). Three of the DAZL piglets (founders1655-1657) were stillborn. Of the six piglets from APC-modified cells,one was stillborn, three died within 1 wk, and another died after 3 wk,leaving only founder 1661 alive. The lack of correlation betweengenotype and survival suggests that the early deaths were related tocloning rather than to gene edits. All six APC piglets were heterozygousfor the intended HDR-edited allele, and all but one piglet had either anin-frame insertion or deletion of 3 bp on the second allele (FIGS. 54 Aand B) in Ossabaw pig fibroblasts. The remaining animals are beingraised for phenotypic analyses of spermatogenesis arrest (DAZL−/−founders) or development of colon cancer (APC+/− founders). Referring toFIG. 6, (a) RFLP analysis of cloned piglets derived from DAZL- andAPC-modified landrace and Ossabaw fibroblasts, respectively. ExpectedRFLP products for DAZL founders are 312, 242, and 70 bp (opentriangles), and those for APC are 310, 221, and 89 bp (filledtriangles). The difference in size of the 312-bp band between WT andDAZL founders reflects the expected deletion alleles. (b) Sequenceanalysis confirming the presence of the HDR allele in three of eightDAZL founders, and in six of six APC founders. BMs in the donortemplates (HDR) are indicated with arrows, and inserted bases areenclosed in blocks. The bold text in the top WT sequence indicates theTALEN-binding sites.

Example 47: GPR54 Knockout

FIG. 55 depicts GPR54 (SEQ ID NO: 196) knockouts, made according to theindicated gene targeting strategy. TALENs designed to bind porcine exon3 (underlined text in SEQ ID NO: 197, 492 and 493) were co-transfectedwith an oligonucleotide homology template (HDR) designed to introduce apremature stop codon (boxed) and a HindIII restriction site. For theexperimental results shown in panel b, 2 micrograms of TALENs encodingmRNA (SEQ ID NO: 519) plus 0.2 nMol (2 uM) of the HDR template weretransfected into pig fibroblasts 500,000 pig fibroblasts using the NEONnucleofection system (Life Technologies) with the following settings: 1pulse, 1800 v; 20 ms width and a 100 ul tip. The cells were grown at 30°C. for three days after exposure to TALENs and cells were enumerated andplated at a range of densities 1-20 cells/cm2 on 10 cm dishes. Cellswere cultured for 10-15 days until individual colonies of 3-4 mm indiameter could be observed. Colonies were aspirated with a p-200pipettor under gentle aspiration and expelled into a well of 24-wellplate with 500 ul of growth medium (Carlson, 2011). Plates with clearlydefined colonies (˜10-30/plate) were chosen for colony aspiration tolimit the chance of aspirating cells from multiple colonies. Once acolony reached 70-90 percent confluent in the 24-well dish, a portionwas harvested for RFLP analysis and the remainder was cryopreserved.panel b) A total of 96 colonies were analyzed for homology dependentrepair by HindIII RFLP assay. DNA from each colony was added to a PCRreaction that included PCR primers flanking the target site; forward(5′-aaggatgtcagcacctctctggg (SEQ ID NO: 159)) and reverse(5′-ACCCACCCGGACTCTACTCCTACCA (SEQ ID NO: 160)). PCR products (389 bp)were added to a HindIII restriction digest and resolved on a 2.5%agarose gel. Each lane represents one colony (see FIG. 81). Cleavageproducts of 231 and 158 bp are indicative of homology dependent repair.Colonies with the parent band of 389 bp are classified as heterozygous(open triangle) and those without are classified as homozygous (filledtriangle) for the HDR, knockout allele. Cells prepared by this techniquewere used to clone pigs using customary techniques (see Example 48).Indeed, GPR54 knockout pigs were born through somatic cell nucleartransfer. Underdeveloped testes were apparent in 6-12 month old malepigs, and such pigs did not exhibit boar taint. Rather, such pigs actedlike barrows, with little to no aggression observed.

Example 48: Creation of Livestock that do not Mature without Treatment

Livestock with GPR54 knockout(s) can be prepared, including cattle, pig,and chicken. The preceding example details one such process. Thefollowing specific methods are described for pigs; artisans will be ableto adapt the experiments to other livestock after reading thisapplication. TALENs for Gpr54 (SEQ ID NO: 196) were developed and usedto generate heterozygous and homozygous knockout cell lines (SEQ ID NO:197, 492 and 493). Analysis of the colony isolation and screening can beseen in FIG. 81. Pregnancy was established using male and femaleGpr54−/− and/or cell lines heterozygous for Gpr54+/− with Gpr54−/−animals generated by intercross. The development and fertility ofGpr54−/− animals was evaluated. The already-demonstrated ability togenerate efficient TALENs, isolate mutant colonies and producetransgenic animals from cells or zygotes has been well documentedherein, see also Tan et al., PNAS, 110(41): 16526-16531, 2013.

Generation of Gpr54−/− male and female pigs. Ten bi-allelic KO male andfemale clones, as generated in Example 10 through somatic cell nucleartransfer (SCNT), harboring frame shift mutations of both alleles werepooled for cloning by SCNT. Two rounds of cloning (3 transfers each)were conducted. Three transfers were performed and two sows wereimpregnated. A total of 19 piglets were born alive and two werestillborn. Genotypes of the resulting animals were characterized bysequencing of the targeted region of Gpr54 as can be seen in FIG. 82.Because Gpr54+/− cells were used for cloning, Gpr54−/− animals weregenerated by intercross.

Phenotypic evaluation of Gpr54−/− pigs. Serum levels of testosterone andFSH (≥3 per sex) were quantified every two weeks for Gpr54−/− animalsand age-matched controls beginning at 5 months and continuing to 9months of age. For males, testicular size was measured and plottedagainst body weight and age. Underdeveloped testes were apparent in 6-12month old male pigs. No boar-taint odor was present and they acted likebarrows in that little to no aggression was observed in the animals.

Example 49: CRISPR/Cas Mediated HDR

CRISPR gRNAs that overlapped the T1591C site of p65 were made andevaluated for introgression in pig fibroblasts (target sequenceidentified in SEQ ID NO: 372). Efficient production of double strandedbreaks (DSBs) at the intended site was observed. CRISPR/Cas9-mediatedHDR was <6% at day 3 and below the limit of detection at day 10.Recovery of modified clones was lower with CRISPR-mediated HDR than withTALENs, even though the TALENs cut 35 bp away from the SNP site (Table7). Analysis of CRISPR/Cas9-induced targeting at a second locus,sAPC14.2, was more efficient, although it did not reach the level of HDRinduced by TALENs at this site (˜30% vs. 60%). See also, Tan et al.,PNAS, 110(41): 16526-16531, 2013). The CRISPR/Cas9 endonucleases weregenerated based on the Church laboratory system and methods, Mali P, etal. (2013) RNA-guided human genome engineering via Cas9. Science339(6121):823-826.

TABLE 11TALEN Sequences (left) and DNA Target Sequences for CRISPR (right).Table 11. TALEN sequences and DNA target sequence of the TALENsDNA Target sequence, spacer underlined TALEN pair TALEN RVD sequence(Sense strand) ssLDLR2.1 HD NG HD HD NG NI HD NI NI NN NG NN NN NICTCCTACAAGTGGATTTGTGATGGGAACACCGAGTGC NG NG NGAAGGACGGGTCCG SEQ ID NO: 354 HD NN NN NI HD HD HD NN NG HD HD NG NGNN HD NI HD NG btGDF83.1L + NN NG NN NI NG NN NI NI HD NI HD NG HD HDGTGATGAACACTCCACAGAATCTCGATGCTGTCGTTAC NR NI HD NI NN NI NI NG HD NGCCTCTAACTGTGGATTTTGA SEQ ID NO: 355NG HD NI NI NI NI NG HD HD NI HD NI NN NG NG NI NN NI NN ssDAZL3.1NN NN NI NG NN NI NI NI HD HD NN NI NI NI NGGGATGAAACCGAAATTAGAAGTTTCTTTGCTAGATATG NG GTTCAGTAAAAG SEQ ID NO: 356HD NG NG NG NG NI HD NG NN NI NI HD HD NI NG NI NG ssAPC14.2NN NN NI NI NN NI NI NN NG NI NG HD NI NN HDGGAAGAAGTATCAGCCATTCATCCCTCCCAGGAAGAC HD NI NGAGAAATTCTGGGTC SEQ ID NO: 357 NN NI HD HD HD NI NN NI NI NG NG NG HD NGNN NG ssTp53 NN NN HD NI HD HD HD NN NG NN NG HD HDGGCACCCGTGTCCGCGCCATGGCCATCTACAAGAAGT NN HD NN HDCAGAGTACATG SEQ ID NO: 358 HD NI NG NN NG NI HD NG HD NG NN NI HD NG NGssKissR3.2 NN HD NG HD NG NI HD NG HD NG NI HD HD HDGCTCTACTCTACCCCCTACCAGCCTGGGTGCTGGGCGA HD CTTCATGTGC SEQ ID NO: 359NN HD NI HD NI NG NN NI NI NN NG HD NN HD HD HD NI ssEIF4GI14.1HD HD NN NG HD HD NG NG NG NN HD HD NI NICCGTCCTTTGCCAACCTTGGCCGACCAGCCCTTAGCAA HD HD NG NGCCGTGGGCCCCCA SEQ ID NO: 360 NG NN NN NN NN NN HD HD HD NI HD NN NNNG NG NN HD NG btGGTA9.1 HD NG NN HD NN HD NG HD HD NG NG HD NI NICTGCGCTCCTTCAAAGTGTTTAAGATCAAGCCTGAGAA NI NN NGGAGGTGGCAGGAC SEQ ID NO: 361 NN NG HD HD NG NN HD HD NI HD HD NG HDNG NG HD NG ssRAG2.1 NI HD HD NG NG HD HD NG HD HD NG HD NGACCTTCCTCCTCTCCGCTACCCAGCCACTTGCACATTC HD HD NN HD NGAAAAGCAGCTTAG SEQ ID NO. 362 HD NG NI NI NN HD NG NN HD NG NG NG NG NNNI NI NG ssIL2Rg2.1 HD HD HD NI NI NI NN NN NG NG HD NI NN NGCCCAAAGGTTCAGTGTTTTGTGTTCAATGTTGAGTACA NN NG NG NGTGAATTGCACTTGG SEQ ID NO: 363 HD HD NI NI NN NG NN HD NI NI NG NG HD NING NN NG NI HD NG btGDF83.6-A NN HD NG HD NG NN NN NI NN NI NI NG NI NGGCTCTGGAGAATATGAATTTGTATTTTTGCAAAAGTATNI NG NN NI NN NN NI NG NI HD NG NG NG NG CCTCAT SEQ ID NO: 364btGDF83.6-G NN HD NG HD NG NN NN NI NN NI NI NG NNNGGCTCTGGAGAATGTGAATTTGTATTTTTGCAAAAGTATNI NG NN NI NN NN NI NG NI HD NG NG NG NG CCTCAT SEQ ID NO: 365ssGDF83.6 NI HD NG NN HD NG HD NG NN NN NI NN NI NNACTGCTCTGGAGAGTGTGAATTTGTATTTTTACAAAAA NG TACCCTCAC SEQ ID NO: 366NN NG NN NI NN NN NN NG NI NG NG NG NG NG NN NG btRosa1.2HD NG HD NN HD NI NG NG NN HD HD HD NI HDCTCGCATTGCCCACTGGGTGGGTGCTTAGGTAGGTAGG NG GTGGAGAGAG SEQ ID NO: 367HD NG HD NG HD NG HD HD NI HD HD HD NG NI HD HD NG ssSRY3.2NI NG NI HD NI NG NG NG NG NI HD NI HD NI HDATACATTTTACACACATATATATGAAACTGACAGTATT NI NG NI NGAATGGCCTGAACCT SEQ ID NO: 368 NI NN NN NG NG HD NI NN NN HD HD NI NG NGNI NI NG caFecB6.1 NI HD NI NN NI NN NN NI NN NN HD HD NI NNACAGAGGAGGCCAGCTGGTTCCGAGAGACAGAAATAT HD NG NN NN NG NGATCAGACGGTGTTGATG SEQ ID NO: 369HD NI NG HD NI NI HD NI HD HD NN NG HD NG NN NI NG NI NG caCLPG1.1NN NI NN NI NN HD NN HD NI NN NN NI NI NGGAGAGCGCAGGAATCCAGGCGCAGGGGCCCGAGGGCT HD HD NI NN NNGGGACCACCTGTCAG SEQ ID NO: 370 HD NG NN NI HD NI NN NN NG NN NN NG HD HDHD NI NN HD btHP1.3 NG NG NG HD NG NG NN NN NG NI NN NN HDTTTCTTGGTAGGCTGGTATTCTTGCTCTTTAGATCAAAA NG NN CTCTCTTTTC SEQ ID NO: 371NN NI NI NI NI NN NI NN NI NN NG NG NG NG NN NI NG ssP65_11.1NN HD HD HD HD HD HD HD NI HD NI HD NI NNGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTAC HD NG CCTGAGGCTAT SEQ ID NO: 372NI NG NI NN HD HD NG HD NI NN NN NN NG NI HD NG ssP65.8HD NG HD HD NG HD HD NI NG NG NN HD NNCTCCTCCATTGCGGACATGGACTTCTCAGCCCTTCTGA NN NI GTCAGATC SEQ ID NO: 346NN NI NG HD NG NN NI HD NG HD NI NN NI NI NN

Example 50: Kiss Gene Conservation

Referring to FIG. 56, the structural organization of the kiss gene isconserved and contains two coding exons, one encoding both the signalpeptide and part of the kiss peptin precursor, the other encoding theremainder of the precursor including the kisspeptin-10 sequence. Theposition of the intron on tilapia Kiss mRNA (corresponding cDNA, SEQ IDNO: 376) is indicated by a triangle glyph. The location of the forwardand reverse primers for PCR amplification of the target region (442 bp)and the binding sites for the two engineered pairs of TALENs, Kiss1.1a(SEQ ID NOS: 456, 457) and Kiss1.1b (SEQ ID NOS:458, 459) are indicatedin black and gray boxes. Panel b shows a schematic representation of thetargeted kiss genomic region showing the location of the kisspeptin-10biologically active peptide and each kiss1.1a and 1b TALENs recognitionsites. PCR (442 bp) and qPCR primer pairs (138 bp amplicon) for analysisof indels are shown as well.

Example 51: Kiss and KissR Knockout in Fish

A. Construction of TALEN Expression Vectors

TABLE SHOWING CONSTRUCTIONSense Left TALEN  -  Sense Spacer  -  Antisense Right TALEN Kiss1.1aACAACCCTCTCAGCCTT CGCTTTGGGAAACGCT ACAATGGCTACATTTAC (SEQ ID NO: 161)Kiss1.1bCGCTTTGGGAAACGCTACAAT GGCTACATTTACAGA AGAGCTGTTAAAAGAGCC (SEQ ID NO: 162)KissR E2CCCCTTCACCGCCACCCTTT ACCCCCTCCCTGGATGG ATCTTTGGCAACTTCATGTGC (SEQ ID NO:163)KissR E3CTACCCCCTGAAATCTCTT CGGCACCGAACCCCCA AAGTAGCCATGATTGTCAGC (SEQ ID NO: 164)

TABLE OF PRIMERS USED Target site Primer Name Primer sequence (5′-3′)Experiment KissRE2 QPCRE2 F GCCACTGACATCATCTTCTTG qPCR (112 bp) SEQ IDNO: 165 QPCRE2 R2 GAAACAGAAAGTTGAAGTGG SEQ ID NO: 166 KissRE3 QPCRE3 FTCACCCTGACTGCTATGAGTGGA qPCR (143 bp) SEQ ID sequencing NO: 167QPCRE3 R2 ATGAGTCAGTCGATAATGACACG SEQ ID NO: 168 KissRE2 GKRE2FTTATGCAAAAGAAGAAAGGTG PCR (622 bp) SEQ ID NO: 169 GKRE2RGCAGAGTTCGACCTACTTTCATTG SEQ ID NO: 170 KissRE3 GKRE3FTATACATAGCCCCCATTTTC AGTG PCR (702 bp) SEQ ID NO: 171 GKRE3RGGCAGCAGGTAGGCAGCAA SEQ ID NO: 172 Kiss1.1a KissFGTCCTCTGCATTCAGGAGA ACAG PCR (442 bp) SEQ ID NO: 173 and b KissRCTAAAAGTATTTTATTTACATAGT SEQ ID NO: 174 Kiss1.1a QPCRkissFAGGCAGCTCCTTTGCAATGAT qPCR (138 bp) SEQ ID sequencing NO: 175 QPCRkissRAGAGAAGGGTGAAAACTTTTT SEQ ID NO: 176

B. Talen mRNA Synthesis.

MINIPREP DNA of pT3 Ts-TALEN were digested with 5-10× Units of SacI-highfidelity for 2 hours in a 200-μL, reaction. Restriction digest wastreated with 8-μL RNAsecure (Ambion) and incubate at 60° C. for tenminutes. RNAsecure treated DNA was purified using the MINIELUTE PCRcleanup kit from Qiagen and eluted in 10-μL of RNAase free injectionbuffer (5 mM Tris Cl, pH 7.5; 0.1 mM EDTA). Synthetic mRNA were producedusing the mMESSAGE MACHINE T3 kit (Ambion) using 1 ug of linearizedtemplate and 1.5 hours incubate at 37° C. After 15 minutes treatmentwith Turbo DNAase the mRNA was purified using the Ambion MEGACLEAR kitand eluted 2× with 50-μL of heated H₂O.

C. Microinjection of TALENs Pairs

RNA encoding each TALEN arm were combined and resuspended in nucleasefree water at a concentration of 10-200 ng/μL. 5-20-pL were injectedinto one cell stage tilapia embryos. Injected embryos survival wasmeasured at 6 days post fertilization against a non injected controlgroup. RNA concentration giving a 50% rate of survival was used forrepeat/standard injections to generate Knock outs. To confirm thatinjected embryos died from TALENs induced mutagenesis, deformed embryoswere collected and mutation at the target site was investigated using aQPCR melt profile analysis.

D. Tissue Collection and DNA Extraction of Control and RNA TreatedTilapia.

Six day old RNA treated embryos (deformed) were dechorionatedanesthetized and the yolk sac was removed using a razor blade. Embryonictissue was digestion overnight in lysis buffer; 10 mM Tris, 10 mM EDTA,200 mM NaCl, 0.5% SDS, 100 mg/ml proteinase K and extracted withautomated Research X-tractor, Corbett robotic system using Whatman™unifilter 800, 96 well plates (GE Healthcare, UK). Embryos that survivedmicroinjection and developed normally (from groups with ˜50% survivalrate) were raised to 1 month of age, anaesthetized; fin clipped andplace in individual jars while their fin DNA was analyzed (overnightdigestion in lysis buffer followed by DNA extraction as describedabove). Sperm was stripped from G0 males carrying somatic mutations atthe kiss or kissR loci and gDNA extracted using DNAzol Reagent (LifeTechnolgies) following standard procedure. Extracted DNA was resuspendedin 30 μl of MQ H2O.

E. Identification of Mutation by QPCR

Real-time qPCR was performed ROTOR-GENE RG-3000 REAL TIME PCR SYSTEM(Corbett Research). 6-μL genomic DNA (gDNA) template (diluted at 1ng/μl) was used in a total volume of 15 μL containing 0.4 μMconcentrations each of the forward and reverse primers and 7.5 μL of 2×Brilliant II SYBR GREEN QPCR MASTER MIX (Agilent Technologies). qPCRprimers were designed using DNAstar software (See above: Table ofPrimers Used). The qPCR was performed using 40 cycles of 15 seconds at95° C., 60 seconds at 60° C., followed by melting curve analysis toconfirm the specificity of the assay (67° C. to 97° C.). In thisapproach, to detect the occurrence of a DNA polymorphism at the targetedkiss and kissR loci, short PCR amplicons (approx 100-140 bp) thatinclude the region of interest are generated from a gDNA sample,subjected to temperature-dependent dissociation (melting curve). WhenTALEN-induced polymorphisms are present in the template gDNA,heteroduplex as well as different homoduplex molecules will be formed.The presence of multiple forms of duplex molecules is detected by Meltprofile, showing whether duplex melting acts as a single species or morethan one species. Generally, the symmetry of the melting curve andmelting temperature infers on the homogeneity of the dsDNA sequence andits length. For example, if small insertion or deletions resulting fromrepair of TALENs-induced DSBs by NHEJ are generated then that meltingtemperature will positively correlate to the length of the deletion orinsertion, proportionally to the energy required to break the base-basehydrogen bonding. If multiple forms of duplex molecules are present, thetemperature dependant denaturation will detect together the mostinstable heteroduplex and the most stable homoduplex giving a modified(dissymmetric) melt profile. The Melt analysis is performed bycomparison with reference DNA sample (from non-injected tilapia controlor plasmid containing the genomic region of interest) amplified inparallel with the same master mix reaction. In short, variation in meltprofile distinguishes sequences carrying TALEN induced mutation fromwild type sequence, thus facilitating the screen.

F. Calculating Mutation Rates in Somatic Cells or Germ Cells ofMicroinjected Tilapia and Characterization of TALEN Induced Mutations.

Fish whose somatic or germ cells gDNA produced asymmetric qPCR meltprofiles (candidate mutant) were further analyzed to measure themutagenic frequency. Genomic PCR products containing the target site(442 bp for Kiss and 720 bp for KissR) were obtained from fin-DNA orsperm-DNA. The PCRs were carried out in a 25-μL reaction mixture, whichcontained 120-180 ng template gDNA, 0.1 μl of Platinum Taq DNApolymerase, 0.2 mM dNTPs, 1× Taq DNA polymerase buffer, 2 mM Mg2+, and0.2 μM of each primer. DNA amplification was done under the followingconditions: 95° C. for 5 min, followed by 35 cycles of 94° C. for 30 s,55° C. for 30 s, and 72° C. for 45s, with a final extension at 72° C.for 2 min. The PCR products were cloned into TOPO 2.1 TA vector(Invitrogen), and transformed into competent E. coli cells (ONE SHOT,Top 10F′, Invitrogen). Transformant colonies were randomly picked with asterile pipette tips and transferred directly onto individual qPCRreaction tubes before replating on selective agar media. qPCR wereperformed using primers that span the TALENs target sites of interest(100-140 bp amplicons). QPCR reactions showing specific productamplification were compared against a reference DNA sample control (wildtype sequence) to identify melt profile variants (FIG. 10 panels c andd). DNA mutation rate was calculated as the number of mutant sequences(colonies with variant melt) divided by the total number of sequencesanalyzed multiplied by 100. To visualize the mutations present at thetarget loci, clones representing individual somatic or sperm cells weredisplayed in a Scatter plot of Ct versus Melt temperatures (see FIG. 10panel d for example). In these graphs, each E. Coli colony isrepresented by a data point (x, y), with x representing its Ct and yrepresenting its melt. Individual colonies carrying identical sequencesshould display similar melting temperature. Colonies showing variantmelting temperature were grown overnight and their plasmid extracted andpurified (MINIPREPARATION kit, QIAGEN). The region containing the TALENstarget site were then sequenced using selected primers for the kiss andkissR regions, as indicated. To characterize mutations in F1 and F2fish, the 442 bp and 702 bp amplicons containing the target kiss1.1a andKissRE3 loci were purified on silica-membrane-based spin column(QIAQUICK PCR PURIFICATION KIT, QIAGEN). The purified PCR were directlysequenced using an internal primer (KissRF).

G. Founder Screen

Gametes were stripped from all putative founders and F1 embryos wereproduced from in vitro fertilization with gametes collected from WTstock. 3 weeks post-fertilization, F1 progeny were fin-clipped and heldseparately in individual jar. Fin DNA was extracted as previouslydescribed (see Tissue collection and DNA extraction section above) andadjusted to 1 ng/μ1 using a spectrophotometer NANODROP ND1000). Ingeneral, 10-20 juveniles from each potential founder were screened byQPCR using the melt analysis strategy described above. For sequenceconfirmation, genomic DNAs from single embryo/juvenile were amplifiedand the PCR product submitted to sequencing after purification.

Sequencing chromatography of PCR showing two simultaneous reads areindicative of the presence of indels. The start of the deletion orinsertion typically begins when the sequence read become divergent. Thedual sequences are than carefully analyze to detect unique nucleotidereads (see FIG. 12 panel a). The pattern of unique nucleotide read isthen analyzed against series of artificial single read patternsgenerated from shifting the wild type sequence over itselfincrementally.

H. Mutagenic Potency of Engineered TALENs

Engineered TALENs and synthetic capped mRNA encoding each heterodimericTALENs together was injected at various concentrations from 10 to 250ng/μ1 into 1-cell stage tilapia embryos. We then observed the injectedembryos at 6 days post fertilization (dpf). Embryos injected with lessthan 10 ng of TALENs developed normally while a dose of 200 ng (Kiss1a)and 10 ng (KissRE3) generated up to 50% of dead or deformed embryos.Dose of 250 ng for kiss1.1b and kissRE2 generated less the 30%mortality. On day five, injected embryos were separated between thosethat developed normally from those with morphological deformities. Tocheck for evidence of mutations, genomic DNA was isolated from a pool of3 deformed embryos for each TALENs treated group and from 3 normalembryos from a non injected control group. Genomic DNA was used for QPCRmelt analysis of the target loci. Asymmetric melt profile were found inthe pool of embryos treated with TALENs targeting the kiss1.1a andkissRE3 loci (data not shown) but not in embryos treated with the other2 TALENs pairs.

To confirm the presence of mutation, 20-40 normally developed juvenilesin each group were assayed by QPCR melt analysis. None of the fishinjected with TALEN KissR-E2 and Kiss1.1b mRNA produced variant meltsuggesting that either no mutation were created or that the mutation didnot produce detectable melt variation. Nevertheless, a total of 8 fishproducing variant melt profiles were found, 4 for each kiss1.1a andKissRE3 loci (FIG. 10 panel a and panel b). To confirm that the observedmelt variation results from a mixture of wild-type and NHEJ productswith micro-insertion or deletion at the target site, each target region(442 bp for Kiss and 702 bp for KissR) was amplified in a PCR reaction.The resulting PCR fragments were cloned into Topo TA vectors andtransformant colonies screened by direct real time-PCR. For each fishtested, between 14 and 21 E. Coli transformant colonies were hand-picked(randomly) and added directly (without DNA purification) to the Q-PCRreaction mixture.

Colonies carrying mutated alleles were identified by comparison to thewild-type unmodified sequence. High frequencies of colonies with variantmelt profiles ranging from 50-91% were detected (FIG. 10, panels c andd).

To characterize some of these lesions, the plasmid from clones thatproduced variant amplicons was extracted and the PCR insert wassequenced. Between 4 and 7 clones were sequenced for each TALENs treatedgroup and all but one carried mutated alleles. A total of fourteendifferent somatic mutations in the kiss and kissr genes were detectedfrom all 8 TALENs treated fish (eight at the Kiss1.1a loci and six atKissRE3 loci). Nine different nucleotide deletions, two insertions, andthree combinations of nucleotide insertions and deletions were observed(FIG. 11 panels a and b). A deletion/insertion of as little as 3 bp wasdetectable by RT-PCR melt analysis. It was observed that TALENs inducedmutation occurs multiple times in an RNA treated fish resulting inmosaic somatic mutations (see table below).

It was found that more than 95% of the sequences from colonies showingmelt variation carry a mutation indicating that DNA mutation rate can beapproximated by measuring the frequency of clones producing variantmelt. Thus, the rate of mutation was calculated to be between 35% and91% depending on the fish. This result indicates the highly efficientintroduction of targeted indels at the expected genomic locations.

The table, Summary of the results of somatic mutation screen, showsresults for TALENs-injected tilapia. The second column describes themutant sequences identified in somatic cells, including the sizes of theindels (+, insertion; −, deletion) and the resulting protein sequencemodification are shown inside the parentheses. In the last column, theestimated rate of somatic mutation for each fish was calculated from thefrequency of colonies producing variant melting temperature.

TABLE Summary of the results of somatic mutation screen % of mosaicsomatic mutations (n = total Fish number of reference Mutation typecolonies screened) Kiss17 +10 nt(frame shift/stop); +4 nt 73% (n = 22)(frame shift/stop); Δl2 nt (−4AA) and Δ18 nt (−6AA) Kiss19 Δ12 nt (−4AA)48% (n = 21) Kiss20 Δ16 nt (frame shift/stop); Δ12 nt (−4AA) 91% (n =23) Kiss41 Δ4 nt (frame shift/stop); Δ12 nt 85% (n = 14) (−4AA and F >C); Δ12 nt (−4AA) RE3-1 Δ10 nt (frame shift/stop); Δ7 nt 35% (n = 21)(frame shift/stop) RE3-4 Δ3 nt (frame shift/stop); Δ26 nt 85% (n = 14)(frame shift/stop) RE3-6 Δ5 nt (frame shift/stop); Δ14 nt 63% (n = 16)(frame shift/stop) RE3-11 Δ7 nt (frame shift/stop) 66% (n = 21)

I. Sequence Analysis of TALENs Mutations

Of the different types of nucleotide mutation, five and six caused aframeshift leading to the generation of premature stop codons in thekiss and kissr gene respectively. Also, there was a high frequency of 12nt deletions at the Kiss1.1a loci which occurred independently in all 4TALENs treated fish. This mutation result in the loss of 4 amino acids(AA).

F0 TALENs-mutated tilapia were raised to sexual maturity and their sexeswere determined. To show thatr TALENs treated fish can induce heritablemutations; genomic DNA was extracted from the semen's of eachspermiating animals and screened. The frequency of sperm carryingmutation was determined by the frequency of clones showing variant meltprofiles as previously described. To characterize the sperm associatedlesions, the plasmids from colonies with variant melt was extracted andsequenced. Germline mutation frequency ranging from 50% to 91% wasobserved. Sequences revealed the existence of multiple indels in eachfish germline.

TABLE Sequencing % of mosaic somatic mutations (n = total Male Fishnumber of reference Mutation type colonies screened) Kiss17 Δl2 nt(−4AA) 50% (n = 20) and Δ18 nt (−6AA) Kiss19 Δl2 nt 65% (n = 30) (−4AA);+3 nt (+1AA) Kiss20 Δ16 nt 91% (n = 23) (frame shift/stop); Δl2 nt(−4AA) RE3-4 Not sequenced 88% (n = 18) RE3-6 Not sequenced

J. Analysis of Germ Line Mutations at the Kiss and kissR Loci.

To further demonstrate that Kiss and kissR TALENs effectively inducedmutation in the germ line, the 8 founders were intercrossed withwild-type stocks. All 8 TALENs treated fish were fertile and producedviable clutches of embryos. These progeny were raised and screened forthe presence of mutated alleles. All 8 founders could transmit heritablemutations. The analysis first showed that the fraction of progenycarrying putative mutation ranged between 16% and 90% as gauged by QPCRmelt profile analysis of F1 fin-DNA extracts. As expected, there was apositive correlation between the extent of mosaicism in the TALENstreated parent and the frequency of progeny carrying a mutation.Analysis of selected gene sequences producing deformed melt profile allrevealed a range of induced indel mutations, some of which werepreviously found in somatic tissue of the founders (FIG. 12 panel b).Furthermore, sequencing of F1 fish producing wild type melt all revealedwild type sequences. More than one type of heritable mutation from asingle founder was often observed, suggesting that those mutationsoccurred independently in different germ cells within the same animal.Inherited mutations included deletions ranging in size from 3 to 18 bp(FIG. 12 panel b). In the progeny of all 4 kiss mutant founders, theonly inherited mutations were deletions of 12 nt and 18 nt whichresulted in the loss of four and six AA. Although, those deletions didnot result in frameshift mutations they remove either one or three AA atthe most C-terminal region of the kiss-10 peptide (FIG. 12 panel c).Because this core sequence was found essential and sufficient for theactivation of the kissR signaling pathway throughout vertebrates, thosemutations would produce a loss of function phenotype. Also identifiedwas a frame shift mutation at the kissRE3 loci which was not previouslyisolated in the founder. All frameshift mutations resulted in apremature stop codon removing between 172 AA and up to 215 AA (±7 nt,FIG. 12 panel c) from the C-terminal portion of the KissR protein. Thesemutations, which remove as much as 57% of the protein sequence, willinactivate the gene function. All kiss and kissr mutations identifiedamong the juveniles F1 offspring were viable in the heterozygous state.

TABLE Summary of founder screening results. In the last column of eachtable, the numbers of embryos carrying indel mutations are shown outsideof the parentheses, and the sizes of the indels are shown inside theparentheses. +, insertion; −, deletion. % F1 with putative #F1 Fishmutations (n = sequenced # of reference total number of (Variant +mutants (sex) F1 screened) WT melt) identified Mutation type Kiss17 ( ♂)66% (n = 30) 13 + 2 13 7{Δ12 nt (−4AA)}and 6 {Δ18 nt (−6AA)} Kiss19 ( ♂)49% (n = 37) 10 + 2 10 10 {Δ12 nt (−4AA)} Kiss20 ( ♂) 73% (n = 29) 12 +2 12 12{Δ12 nt (−4AA)} Kiss41( ♀) 16% (n = 38)  6 + 2 6 6{Δ12 nt (−4AA)}RE3-1( ♀) 29% (n = 44) 19 + 2 19 10{Δ3 nt (−1AA, R > Q); 8 {Δ11 nt(frame shift/stop)}; 1{ Δ8 nt, (frameshift/stop)} RE3-4 90% (n = 22)10 + 2 10 9{Δ9 nt (−3AA)}; ( ♂) 1{Δ5 nt, (frame shift/stop)} RE3-6 ( ♂)RE3-11 63% (n = 35) 11 + 2 11 10{ Δ7 nt ( ♀) (frame shift/stop); 1{Δ5 nt(frame shift/stop)}

K. F1 and F2 Generations

F1 heterozygous mutants showed no morphological defect as they continuedto develop, and all differentiated into fertile adult of both sex. Theabsence of a reproductive phenotype in sexually mature F1 generation isnot unexpected given the presence of a wild type allele of each targetedgene in all somatic cells of selected mutant. The characterization of aninactivation phenotype is only possible in the F2 generation in fishcarrying the associated loss-of-function mutation in the homozygous (orcompound heterozygous) state. To generate homozygous mutation, sperm andeggs collected from F1 heterozygous mutant were used to produce F2generations.

Example 52: Introgression of the Bovine Polled Allele into Horned Cellsby TALEN Stimulated HR

The polled allele schematic is shown in FIG. 61. Four TALEN pairs weredesigned to cut 3′ of the region duplicated in polled (FIG. 61). Thetarget sequences of the TALENs are shown in FIG. 61B (HP1.1 left andright (SEQ ID NOs: 240 and 347); HP1.2 left and right (SEQ ID NOS: 348and 149); HP1.3 left and right (SEQ ID NOS: 150 and 151); HP1.4 left andright (SEQ ID NOS: 152 and 153). The TALEN sequences are provided asfollows (HP1.1 left and right (SEQ ID NOs: 460 and 461); HP1.2 left andright (SEQ ID NOS: 462 and 463); HP1.3 left and right (SEQ ID NOS: 464and 465); HP1.4 left and right (SEQ ID NOS: 466 and 467). HornedHolstein fibroblasts were transfected with mRNA encoding the TALEN pairsand analyzed for activity 3 days post transfection. Surveyor assayrevealed activity of each TALEN pair (FIG. 61). Peak activity wasobserved with HP1.3 and thus was chosen for subsequent experiments.Horned Holstein primary fibroblasts were transfected with 2 microgramsof HP1.3 TALEN mRNA along with ssDNA repair templates at the indicatedquantities and treatments (FIG. 64). One of the repair templates isshown schematically in FIG. 62a (1594 bp template, SEQ ID NO: 381) andis a contiguous sequence from Angus genomic DNA containing a duplicationof 212 bp that replaces a 10 bp deletion relative to the horned allele.The 212 bp duplication is depicted by arrows in both FIGS. 61a and 62a ,the deletion of 10 bp is depicted by a short arrow in FIG. 61a . The 5′homology arm can be defined as the 3′ section of the 1594 bp templatebeginning immediately after the 212 bp duplication (a total of 555 bp).Hence 1594 subtract 827 subtract 555=212, the length of the insertedsequence/duplication that comprises the Celtic polled allele. Beingidentical to the exogenous Angus allele, the TALENs can no longereffectively cleave the polled allele due to separation by about 212 bpbecause of the 212 duplication. Populations of cells three days posttransfection were analyzed for conversion to polled by PCR. Coating ofthe repair template with NLS-RecA-Gal4 (Liao and Essner 2011) had asignificant effect on the frequency of polled conversion (FIG. 64 panelsb and c). Polled conversion was also apparent in individual colonies(FIG. 63).

Methods: Approximately 600,000 cells were transfected with the NEONtransfection system under the following parameters (1 pulse; 1800 v; 20ms width). Each transfection consisted to two micrograms of TALEN mRNAalong with the indicated repair template. Repair template was coatedwith Gal4:RecA by the following method. Five hundred nanograms (3 ultotal) of repair template PCR product was incubated for 10 min at 95° C.and placed on ice for 2 minutes prior to addition of 0.8 ul of buffer[100 mM Tris OAc, pH 7.5; 500 mM NaOAc; 10 mM DTT; 10 mM Mg(OAc)2], 0.6ul 16.2 mM ATPγS (Sigma) and 1,250 ng of NLS-RecA-Gal4 in a totalreaction volume of 8 ul. This reaction was then incubated at 37° C. for30 minutes and placed on ice. The entire volume was used in a singletransfection. Cells were cultured and analyzed using methods describedin Carlson, Tan et al. 2012. The 591 bp HDR template was used.

Example 53

Cells made by, or embryos modified by, the methods described herein tointrogress polled alleles are cloned and/or placed in surrogate females,gestated, and born as live animals comprising the POLLED allele. Wholelive animals made from the cells of Example 22B are shown in FIG. 68.The animals shown do not have horns and are healthy animals that areHolsteins with a non-meiotically introgressed pollele allele thatreplaces the cognate portion of the native horned allele in Holsteincells. The conversion is confirmed by PCR as shown in FIG. 62B.

Example 54

Porcine cells were modified with CRISPR/Cas9 nuclease to knockout theKISS1 gene (SEQ ID NO: 374) in porcine cells by homology dependentrepair (HDR) using sgRNA ssKiss1 c.2.9 (SEQ ID NO: 391). FIG. 73 showsthe CRISPR/Cas9 target sequence, ssKiss1 Exon 2 (SEQ ID NO: 389), andthe repair template, ssKISS1 Ex2.9 (SEQ ID NO: 393) designed to beinserted by HDR within ssKiss1 Exon 2. The alteration effectivelycreates a premature termination codon followed by a HindIII restrictionsite for restriction fragment length polymorphism (RFLP) genotyping (SEQID NO: 394). As a control, a repair template containing a cut blockingmutation, ssKiss1c.2.9 Blocking HDR (SEQ ID NO: 390) was co-injectedwith the ssKiss1 Ex2.9 to increase the chance of heterozygous knockoutoffspring versus homozygous knockout offspring. FIG. 74 shows theCRISPR/Cas9 target sequence, ssKiss1 Exon 2, and the repair templatessKiss1c.2.9 Blocking HDR designed to be inserted by HDR within ssKiss1Exon 2. FIG. 75 shows the results of RFLP analysis of porcine cellsinjected with the ssKiss1c.2.9 Blocking HDR oligonucleotide, generatinga AcuI restriction site.

Example 55

Male pig fibroblasts were either transfected (+, transf.) ornon-transfected controls (C, untransf.). Cells were transfected with acombination of IDT Alt-R crRNA:Tracer RNA complex, Cas9=Alt-R HiFi Cas9nuclease (protein) and ssKiss1 c.2.9 HD3 HDR (SEQ ID NO: 394). FIG. 76,panel A show RFLP analysis results 3 days after transfection. Female pigfibroblasts were transfected with IDT Alt-R crRNA:Tracer RNA complex,Cas9=Alt-R HiFi Cas9 nuclease (protein) with and without the ssKiss1c.2.9 HD3 HDR template. FIG. 76, panel B shows RFLP analysis results 3days after transfection. Cells from these populations were plated at lowdensity for isolation of single cell derived colonies (next slide) andevaluated for editing. Select homozygous HDR clones were confirmed bySanger Sequencing and used for cloning founder animals. FIG. 77 showsRFLP analysis results of individual colonies propagated from thetransfected populations, resulting in three outcomes: Mutant RFLP (2),Heterozygous RFLP (3), or wild type (WT) RFLP(1). FIG. 78 shows theresults of Sanger Sequencing of RFLP positive colonies.

Example 56

Transfection of pig fibroblasts to knockout Kiss1 by HDR. The injectedzygotes were injected with 25 ng/μl gRNA; 50 ng/μl Cas9; 33.3 ng/μl HD3HDR; 66.7 ng/μl Blocking HDR. There were a total of 24 blastocystsresulting from injection. 18 of the 24 samples were subjected to wholegenome amplification, PCR over the target site, and Sanger sequencing.Amplicons were sequenced using Sanger sequencing followed by analysisusing ICE software, Synthego. 61% of the blastocysts were wild type(WT), 39% had a mutation within >10% of the allele. HDR was successfulin one blasocyst, sequencing data show that it was repaired with theBlocking Oligo and was mono-allelic. FIG. 79 shows the results, ˜22% ofinjected zygotes were heterozygous, meaning that they were 31-60% mutantcells.

Example 57

Transfection of pig fibroblasts to knockout Kiss1 by HDR. Similar toExample 56, zygotes were injected with 25 ng/μl gRNA; 25 ng/μl Cas9;26.7 ng/μl HD3 HDR; 53.3 ng/μl Blocking HDR. There were a total of 14blastocysts resulting from injection. 11 of the 14 samples weresubjected to whole genome amplification, PCR over the target site, andSanger sequencing. 36.4% were wild type and the rest had biallelicmutation or monoallelic mutation. Five embryos had HDR events. Oneembryo was mono-allelic for the HD3 HDR, one embryo was mono-allelic forboth HDR templates (HD3 HDR and Blocking HDR) and three embryos werepositive for Blocking HDR (one of which was bi-allelic). FIG. 80 showsthe results. The embryos were implanted in a sow, resulting in a broodof piglets. Nineteen piglets were born and two were born stillborn.

Example 58

Cells can made by, or embryos modified by, the methods described inExample 51 to knockout KISS 1 in bovine cells (SEQ ID NO: 375).

TABLE 8 TALEN to Figure mapping Table 8: TALEN to Figure mapping. TargetRepair TALENs HDR FIG. identified Animal Gene type form Cell type TALENID template Samples (n=)  3B no Bovine ACAN NHEJ mRNA Embryo btACAN12 —6  3C no Porcine p65 NHEJ mRNA Embryo ssP65_11-1 — 14   4A yes BovineACAN NHEJ mRNA Embryo btACAN12 — 4  4B yes Porcine p65 NHEJ mRNA EmbryossP65_11-1 — 17   5B no Porcine DMD NHEJ Plasmid Fibroblast ssDMDE7.1 —Population no Bovine ACAN NHEJ Plasmid Fibroblast btACAN12 — Population 5C no Porcine DMD NHEJ Plasmid Fibroblast ssDMDE7 — Population noPorcine LDLR NHEJ Plasmid Fibroblast ssLDLR4.1 — Population no BovinePRNP NHEJ Plasmid Fibroblast btPRNP3.1 — Population no Porcine GDF8 NHEJPlasmid Fibroblast ssGDF83.2 — Population  6 yes Bovine ACAN NHEJPlasmid Fibroblast btACAN12 — 12   7B, C No Bovine GDF8 NHEJ PlasmidFibroblast btGDF83.1 — Population No Bovine ACAN NHEJ Plasmid FibroblastbtACAN12 — Population No Porcine DMD NHEJ Plasmid Fibroblast ssDMDE7.1 —Population No Porcine DMD NHEJ Plasmid Fibroblast ssDMDE6 — PopulationNo Porcine LDLR NHEJ Plasmid Fibroblast ssLDLR2.1 — Population  8A yesPorcine LDLR NHEJ Plasmid Fibroblast ssLDLR2.1 — 5  8B yes-8A PorcineLDLR NHEJ Plasmid Fibroblast ssLDLR2.1 — 5  9A yes Porcine DMD NHEJPlasmid Fibroblast ssDMDE7.1 — 8  9B yes Porcine LDLR NHEJ PlasmidFibroblast ssLDLR2.1 — 10  10 yes Porcine DMD NHEJ/ Plasmid FibroblastssDMDE6 — Population large deletion yes Porcine DMD NHEJ/ PlasmidFibroblast ssDMDE7.1 — Population large deletion 11 yes Porcine DMDNHEJ/ Plasmid Fibroblast ssDMDE6 — 14  large deletion yes Porcine DMDNHEJ/ Plasmid Fibroblast ssDMDE7.1 — Same As large Above deletion 12 yesPorcine DMD NHEJ/ Plasmid Fibroblast ssDMDE6 — 8 inversion yes PorcineDMD NHEJ/ Plasmid Fibroblast ssDMDE7.1 — Same As inversion Above 13 yesBovine GDF8 HDR Plasmid Fibroblast btGDF83.1 dsDNA- Population 1623 bp(BB-HDR) 14 yes FIG. 13 Bovine GDF8 HDR Plasmid Fibroblast btGDF83.1dsDNA- 4 1623 bp (BB-HDR) 15 yes Porcine LDLR HDR Plasmid FibroblastssLDLR4.2 dsDNA, Population LdlrE4N- stop 16 Yes. FIG. 15 Porcine LDLRHDR Plasmid Fibroblast ssLDLR4.2 dsDNA, 8 LdIrE4N- stop 18 Yes BovineGDF8 HDR Plasmid Fibroblasts btGDF83.1 AAV-1623 Population bp (BB- HDR)19 Yes Bovine GDF8 HDR Plasmid Fibroblasts btGDF83.1 Oligos; BB-Population HDR sense, BB-HDR antisense 20A Yes-4B Porcine p65 NHEJ mRNAFibroblasts ssP65_11-1 — Population or Modified mRNA 20B Yes-9A PorcineDMD NHEJ mRNA Fibroblast ssDMDE7.1 — Population or Modified mRNA 21Yes-13 Bovine GDF8 HDR Plasmid Fibroblasts btGDF83.1 Oligos; BB-Population or HDR sense and colonies mRNA 72 22 Yes Bovine GDF8 HDR mRNAFibroblasts btGDF83.6 Oligos; seq Population ID 135 23 Yes Bovine GDF8HDR mRNA Fibroblasts btGDF83.6 Oligos; seq Population ID 135 24 NoPorcine GDF8 HDR mRNA Fibroblasts ssGDF83.6 Oligos; Seq Population ID146 25 Yes Porcine LDLR NHEJ mRNA Fibroblast ssLDLR2.1 Oligos; SeqPopulation ID 137 and colonies 184 26 Yes-9A Porcine DMD NHEJ PlasmidGermline ssDMDE7.1 — Population or stem cells mRNA 27A Yes Chicken DDX4NHEJ Plasmid DF1 cells ggVASA1.1 — Population VASA Yes Chicken DDX4 NHEJPlasmid DF1 cells ggVASA7.1 — Population VASA 27C Yes Chicken DDX4 HDRPlasmid Primordial ggVASA1.1 dsDNA- Population VASA germ cells Donortargeting vector “Population” = to targeted modification of 1,000-20,000cells in the experimental group

TABLE 9 TALEN to Table mapping Table 9: TALEN to Table mapping. TargetRepair TALENs HDR Samples Table identified Animal Gene type form Celltype TALEN ID template (n=) 1 Yes-FIG. 8A Porcine LDLR NHEJ PlasmidFibroblast ssLDLR2.1 — 275 Yes-FIG. 15 Porcine LDLR NHEJ PlasmidFibroblast ssLDLR4.2 — 95 Yes-FIG. 10 Porcine DMD NHEJ PlasmidFibroblast ssDMDE6 — 35 Yes-FIG. 9A Porcine DMD NHEJ Plasmid FibroblastssDMDE7.1 — 70 no Porcine GHRHR NHEJ Plasmid Fibroblast ssGHRHR2.3 43Yes-FIG. 6 Bovine ACAN NHEJ Plasmid Fibroblast btACAN12 — 35 Yes-FIG. 13Bovine GDF8 NHEJ Plasmid Fibroblast btGDF83.1 — 29 2 no Bovine ACAN NHEJmRNA Embryo btACAN11 — 154 Yes-FIG. 6 Bovine ACAN NHEJ mRNA EmbryobtACAN12 — 227 no Bovine PRNP NHEJ mRNA Embryo btPRNP3.2 — 115 Yes-FIG.13 Bovine GDF8 NHEJ mRNA Embryo btGDF83.1 — 115 3 Yes-FIG. 13 BovineGDF8 NHEJ mRNA Embryo btGDF83.1 — 2 embryos 4 yes Porcine LDLR NHEJPlasmid Pig tail ssLDLR2.1 — 22 biopsy 5 Rows 1-12 — same as Table 1. noPorcine GHRHR NHEJ mRNA Fibroblast ssGHRHR2.3 38 Yes-FIG. 8A PorcineLDLR NHEJ mRNA Fibroblast ssLDLR2.1 — 166 Yes-FIG. 13 Bovine GDF8 NHEJmRNA Fibroblast btGDF83.1 — 86

TABLE 10 TALEN RVD Codes Table 10: TALEN RVD codes TALEN ID TALEN RVD 5′(Left) TALEN RVD 3′ (Right) Target Sequence btACAN12HD HD NG NG NG HD HD NG HD NN HD NG HD HD NG HD NG NN SEQ ID NO: 1HD NI NN NN NN NI NG HD HD HD NG NG NN HD NG NG HD NG HD NG HD NI NN NGbtGDF83.1 NN NG NN NI NG NN NI NI HD NI NG HD NI NI NI NI NG HD HD NI HDSEQ ID NO: 116 HD NG HD HD NI HD NI NN NI NI NI NN NG NG NI NN NI NNNG HD NG btGDF83.2 NN NG NN NI NG NN NI NI HD NING HD NI NI NI NI NG HD HD NI HD HD NG HD HD NI HD NI NN NI NINI NN NG NG NI NN NI NN NG HD NG btPRNP3.1 NN HD NI NI NN NI NI NN HD NNNI NG HD NN NN HD NG HD HD HD NI HD HD NI NI NI NI HD HD NGHD HD HD NI NN NG ssDMDE6 HD NG NI NG NI HD HD NG NI NNNI NN NG NG NG NN NG NG NN HD SEQ ID NO: 91 NN NG HD NI NI NI NI NI NGNI NI NG HD HD NI NN HD HD NI NG ssDMDE7 HD NN HD HD NI NI NN NG NI NGHD HD HD NI NI NI NI NG NN HD NI HD NI NN NG NG NI NN NN HD NIHD NG NI NI HD HD NG NG SSDMDE7.1 NN NN NI NI HD NI NG NN HD NIHD HD NI NN NG NI NN NG NG NG SEQ ID NO: 55 NG NG HD NI NI HD NI NGHD NG HD NG NI NG NN HD HD NG ssLDLR2.1 HD NG HD HD NG NI HD NI NI NNHD NN NN NI HD HD HD NN NG HD SEQ ID NO: 39 NG NN NN NI NG NG NGHD NG NG NN HD NI HD NG ssLDLR4.1 HD HD NI HD NG HD HD NI NN HDHD NN NG HD NI NN NI HD NG NG NG NN NN HD NN HD NGNN NG HD HD NG NG NN HD NI NN NG ssGDF83.2 HD NG NI NI HD NG NN NG NNHD NG NG NG NG NN NN NN NG NN NI NG NG NG NG NN NI NI NNNN HD NI NI NG NI NI NG HD NG ssLDLR4.2 HD HD NI NN NG NN HD NI NI HDHD NI NN NN NI NG HD NI HD HD NI NN HD NG HD HD NI HD HD NGNI NG HD NI HD NI NN NN HD HD ssP65_11-1 NN HD HD HD HD HD HD HD NINI NG NI NN HD HD NG HD NI NN SEQ ID NO: 7 HD NI HD NI NN HD NGNN NN NG NI HD NG btGDF83.6 NN HD NG HD NG NN NN NI NNNI NG NN NI NN NN NI NG NI HD SEQ ID NO: 135 NI NI NG NN NG NG NG NG NGssGDF83.6 NI HD NG NN HD NG HD NG NN NN NG NN NI NN NN NN NG NI NGNN NI NN NI NN NG NG NG NG NG NN NG ggVASA1.1NN HD NG NI NI HD NN NG NN HD HD HD NG HD HD NG HD HD NI NGNG HD HD NG NN NN NG HD HD NI NN HD NN NI NI NG NG ggVASA7.1NI NG NI NG HD NG NI NI NI NI HD NN HD NG NN NG NN HD NG NGNG NN NN NI NG HD NG NN HD NI HD NG ssGHRHR2.3HD HD HD HD NG NN HD HD HD HD HD HD HD HD NG HD NI HD HDNN NN HD NG NG NG HD NG NG NG NN NN HD NG HD NG btACAN11NN HD NI NI NG HD HD HD NI NN NN NG NI NN NN HD NI NI NN NGNN HD NG NG HD NI HD HD NN NG HD HD HD NI NG NG HD HD NN NG NG NGbtPNRP3.2 HD NI NI NG NN NN NI NI HD NI NI NN HD NI NN HD NI NN HD NG HDNI HD HD HD NI NN NG NI NI HD NG NN HD HD NI HD NI NG NN HD NG NGssP65_11-1 NN HD HD HD HD HD HD HD NI NI NG NI NN HD HD NG HD NI NNHD NI HD NI NN HD NG NN NN NG NI HD NG btGDF83.6NN HD NG HD NG NN NN NI NN NI NG NN NI NN NN NI NG NI HD NI NI NG NI NGNG NG NG NG ssGDF83.6 NI HD NG NN HD NG HD NG NNNN NG NN NI NN NN NN NG NI NG NN NI NN NI NN NG NG NG NG NG NN NGggVASA1.1 NN HD NG NI NI HD NN NG NN HD HD HD NG HD HD NG HD HD NI NGNG HD HD NG NN NN NG HD HD NI NN HD NN NI NI NG NG ggVASA7.1NI NG NI NG HD NG NI NI NI NI HD NN HD NG NN NG NN HD NG NGNG NN NN NI NG HD NG NN HD NI HD NG ssGHRHR2.3HD HD HD HD NG NN HD HD HD HD HD HD HD HD NG HD NI HD HDNN NN HD NG NG NG HD NG NG NG NN NN HD NG HD NG ss btPNRP3.2HD NI NI NG NN NN NI NI HD NI NI NN HD NI NN HD NI NN HD NG HDNI HD HD HD NI NN NG NI NI HD NG NN HD HD NI HD NI NG NN HD NG NGssDAZL3.1 NN NN NI NG NN NI NI NI HD HD HD NG NG NG NG NI HD NG NN NINN NI NI NI NG NG NI HD HD NI NG NI NG ssTp53 NN NN HD NI HD HD HD NN NGHD NI NG NN NG NI HD NG HD NG NN NG HD HD NN HD NN HD NN NI HD NG NGssAPC14.2 NN NN NI NI NN NI NI NN NG NI NN NI HD HD HD NI NN NI NI NGNG HD NI NN HD HD NI NG NG NG HD NG NN NG ssKISSRNN HD NG HD NG NI HD NG HD NN HD NI HD NI NG NN NI NI NNNG NI HD HD HD HD NG HD NN HD HD HD NI ssIL2RG2.1HD HD HD NI NI NI NN NN NG NG HD HD NI NI NN NG NN HD NI NIHD NI NN NG NN NG NG NG NG NG HD NI NG NN NG NI HD NG btGDF83.6NN HD NG HD NG NN NN NI NN NI NG NN NI NN NN NI NG NI HD NI NI NG NI NGNG NG NG NG ssRAG2.1 NI HD HD NG NG HD HD NG HDHD NG NI NI NN HD NG NN HD NG HD NG HD NG HD HD NN HD NGNG NG NG NN NI NI NG btGGTA9.1 HD NG NN HD NN HD NG HD HDNN NG HD HD NG NN HD HD NI HD NG NG HD NI NI NI NN NGHD NG HD NG NG HD NG Kiss1.1a NI HD NI NI HD HD HD NG HD NGNN NG NI NI NI NG NN NG NI NN (Tilapia) HD NI NN HD HD NG NGHD HD NI NG NG NN NG Kis1.1b HD NN HD NG NG NG NN NN NNNN NN HD NG HD NG NG NG NG NI (tilapia) NI NI NI HD NN HD NG NI HD NINI HD NI NN HD NG HD NG NI NG ssKISSR3.2 NN HD NG HD NG NI HD NG HDNN HD NI HD NI NG NN NI NI NN NG NI HD HD HD HD NG HD NN HD HD HD NIssElF4G114.1 HD HD NN NG HD HD NG NG NG NG NN NN NN NN NN HD HD HD NINN HD HD NI NI HD HD NG NG HD NN NN NG NG NN HD NG btHP1.1NN NI NN NI NG NI NN NG NG NG NN NI NI NI NI NN NI NN NI NN NGNG HD NG NG NN NN NG NG NG NG NN NI NG HD NG NI NI NI btHP1.2NI NN NG NG NG NG HD NG NG NN NI NI NI NI NN NI NN NI NN NGNN NN NG NI NN NN NG NG NG NN NI NG HD NG NI btHP1.3NG NG NG HD NG NG NN NN NG NN NI NI NI NI NN NI NN NI NN NGNI NN NN HD NG NN NG NG NG NN NI NG btHP1.4 NG HD NG NG NN NN NG NI NNNN NI NI NI NI NN NI NN NI NN NG NN HD NG NN NN NG NG NG NG NN btPRLR9.1NN NN HD HD NN NN HD NI HD NG NI NI NI NN HD NI NG NN NG (SLICK TRAIT)HD NI HD NI NN HD HD NG NN NN NG HD NG NN NG caCLPG1.1NN NI NN NI NN HD NN HD NI NN NN NI HD NI NN NN NG NN NN NGNN NI NI NG HD HD NI HD HD HD NI NN HD HD caCLPG1.1a Same as aboveNI NI HD NI NN NN NG NN NN NG HD HD HD NI NN HD HD caCLPG1.1bSame as above NI NG HD NI NN NN NG NN NN NG HD HD HD NI NN HD HDcaCLPG1.1c Same as above NI NG NI NI NN NN NG NN NN NGHD HD HD NI NN HD HD btDGAT14.2 NN NN HD NI NN NN NG NI NI NNNI NN HD NG HD NI HD NN NN NG NN HD NN NN HD HD NN HD NN HD NGbtDGAT14.4 HD NN HD NI NN NN NG NI NI NN Same as above NN HD NN NN HD HDbtDGAT14.5 NN NN NI NI NN NN NG NI NI NN Same as above NN HD NN NN HD HDbtDGAT14.6 NN NN HD NI HD NN NG NI NI NN Same as above NN HD NN NN HD HD

TABLE 13 TALEN Sequences TALEN ID Left SEQ ID NO: Right SEQ ID NO:ACAN11 +263 399 ACAN11 +231 400 ACAN12 +231 401 402 ACAN12 + 63 403 404DMDE7 +63 406 407 DMDE7.1 +231 408 409 DMDE7.1 +63 410 411 DMDE7 +231412 413 LDLR 4.1 +231 414 415 LDLR 4.1 +63 416 417 btPRNP3.1 +231 418419 btPRNP3.1 +63 420 421 ssGDF83.2 +231 422 423 ssGDF83.2 +63 424 425btGDF8 (also 428 431 btGDF83.1) +63 btGDF8 (also 520 521 btGDF83.1) +231DMDE6 +231 434 437

TABLE 14 full plasmid sequence-pT3Ts-GoldyTALEN (+63 scaffold) TALEN IDLeft SEQ ID NO: Right SEQ ID NO: LDLR2.1 +63 438 439 ssP65_11-1 440 441btGDF 83.6 +63 442 443 ssGDF 83.6 +63 444 445 ggVASA1.1 446 447ggVASA7.1 448 449 btPNRP3.2 450 451 ssTp53 452 453 ssKISS 454 455KISS1.1a (Tilapia) 456 457 KISS1.1 b 458 459 (Tilapia) btHP1.1 460 461btHP1.2 462 463 btHP1.3 464 465 btHP1.4 466 467 btPRLR9.1 (Slick 468 469trait) caCLPG1.1 470 471 caCLPG1.1a 472 473 caCLPG1.1b 474 475 caCLPG1.1c 476 477 ssGRHRH 2.3 +63 478 479 ssDAZLe 3.1 +63 480 481 ssAPC14.2 +63482 483 ssIL2RG 2.1 +63 484 485 btGDF83.6 +63 486 487 ssRAG2.1 +63 488489 btGGTA 9.1 +63 490 491 ssKISSR3.2 +63 492 493 ssEIF4GI 14.1 +63 494495 btDGAT14.2 +63 496 497 btDGAT14.4 +63 498 btDGAT14.5 +63 499btDGAT14.6 +63 500

REFERENCES

Patent applications, patents, publications, and journal articles setforth herein are hereby incorporated herein by reference for allpurposes; in case of conflict, the specification is controlling.

-   A. M. Geurts, et al., Knockout rats via embryo microinjection of    zinc-finger nucleases, 325 Science (2009).-   B. Reiss, et al., RecA protein stimulates homologous recombination    in plants, 93 Proc Natl Acad Sci USA (1996).-   B. Reiss, et al., RecA stimulates sister chromatid exchange and the    fidelity of double-strand break repair, but not gene targeting, in    plants transformed by Agrobacterium, 97 Proc Natl Acad Sci USA    (2000).-   C. Mussolino, et al., A novel TALE nuclease scaffold enables high    genome editing activity in combination with low toxicity, Nucleic    Acids Res (2011).-   Carlson et al., (2013) “Efficient nonmeiotic allele introgression in    livestock using custom endonulceases.” Proceedings of the National    Academy of Sciences.-   Carlson, D. F., W. Tan, et al. (2012). “Efficient TALEN-mediated    gene knockout in livestock.” Proceedings of the National Academy of    Sciences.-   D. A. McGrew & K. L. Knight, Molecular design and functional    organization of the RecA protein, 38 Crit Rev Biochem Mol Biol    (2003).-   D. F. Carlson, et al., Strategies for selection marker free swine    transgenesis using the Sleeping Beauty transposon system, 20    Transgenic Res (2011).-   D. J. Blake, et al., Function and genetics of dystrophin and    dystrophin-related proteins in muscle, 82 Physiol Rev (2002).-   D. Y. Guschin, et al., A rapid and general assay for monitoring    endogenous gene modification, 649 Methods Mol Biol (2010).-   E. E. Perez, et al., Establishment of HIV-1 resistance in CD4+ T    cells by genome editing using zinc-finger nucleases, 26 Nat    Biotechnol (2008).-   H. J. Lee, et al., Targeted chromosomal deletions in human cells    using zinc finger nucleases, 20 Genome Res (2010).-   H. J. Kim, et al., Targeted genome editing in human cells with zinc    finger nucleases constructed via modular assembly, 19 Genome Res.    (2009).-   I.D. Carbery, et al., Targeted genome modification in mice using    zinc-finger nucleases, 186 Genetics (2010).-   J. C. Miller, et al., A TALE nuclease architecture for efficient    genome editing, 29 Nature Biotech. (2011).-   L. Grobet, et al., A deletion in the bovine myostatin gene causes    the double-muscled phenotype in cattle, 17 Nat Genet (1997).-   L. Tesson, et al., Knockout rats generated by embryo microinjection    of TALENs, 29 Nat Biotechnol (2011).-   Liao, H. K. and J. J. Essner (2011). “Use of RecA fusion proteins to    induce genomic modifications in zebrafish.” Nucleic acids research    39(10): 4166-4179.-   M. Christian, et al., Targeting DNA double-strand breaks with TAL    effector nucleases, 186 Genetics (2010).-   M. M. Cox, Recombinational DNA repair in bacteria and the RecA    protein, 63 Prog Nucleic Acid Res Mol Biol (1999).-   Medugorac, I., D. Seichter, et al. (2012). “Bovine polledness—an    autosomal dominant trait with allelic heterogeneity.” PloS one 7(6):    e39477.-   N. Takahashi & I. B. Dawid, Characterization of zebrafish Rad52 and    replication protein A for oligonucleotide-mediated mutagenesis, 33    Nucleic Acids Res (2005).-   O. G. Shcherbakova, et al., Overexpression of bacterial RecA protein    stimulates homologous recombination in somatic mammalian cells, 459    Mutat Res (2000).-   R. J. Yanez & A. C. Porter, Gene targeting is enhanced in human    cells overexpressing hRAD51, 6 Gene Ther (1999).-   R. Kambadur, et al., Mutations in myostatin (GDF8) in double-muscled    Belgian Blue and Piedmontese cattle, 7 Genome Res (1997).-   T. Cermak, et al., Efficient design and assembly of custom TALEN and    other TAL effector-based constructs for DNA targeting, (in press)    Nucl. Acids Res. (2011).-   T. Mashimo, et al., Generation of knockout rats with X-linked severe    combined immunodeficiency (X-SCID) using zinc-finger nucleases, 5    PLoS One (2010).-   Y. Doyon, et al., Transient cold shock enhances zinc-finger    nuclease-mediated gene disruption, 7 Nat Methods (2010).-   Z. Cui, et al., RecA-mediated, targeted mutagenesis in zebrafish, 5    March Biotechnol (NY) (2003).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Lengthy table referenced here US10893667-20210119-T00001 Please refer tothe end of the specification for access instructions.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10893667B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method for producing a livestock animal havinga first allele of a gene of a first livestock breed replaced with asecond allele of the gene, comprising: (a) providing (i) a cell orembryo from a livestock animal of a first livestock breed, wherein thecell or embryo comprises in its genome a first allele of a gene, (ii) aTALEN that specifically binds to and cleaves DNA at a specific site inthe first allele of the gene; and (iii) a homology directed repair (HDR)template comprising a nucleic acid comprising a second allele of thegene, wherein the second allele was identified in a livestock animal ofa second livestock breed; (b) introducing the TALEN and the HDR templateinto the cell or embryo, wherein the TALEN creates a double-strandedbreak in the specific site and the HDR template inserts into thedouble-stranded break, thereby inserting the second allele of the geneinto the genome of the cell or embryo, thereby replacing the firstallele of the gene with the second allele of the gene; and (c) (i)forming a nuclear transfer embryo from the cell of step (b) andtransferring it into a surrogate female or (ii) transferring the embryoof step (b) into a surrogate female to produce a livestock animal havinga first allele of a gene of a first livestock breed replaced with asecond allele of the gene, wherein the second allele was identified in alivestock animal of the second livestock breed.
 2. The method of claim1, wherein the second allele of the gene is linked to a trait of thesecond livestock breed.
 3. The method of claim 1, wherein the TALENcomprises a left TALEN and a right TALEN.
 4. The method of claim 1,wherein the livestock animal of the first livestock breed is chosen fromthe group consisting of swine, cow, sheep, goat, chicken, rabbit, andfish.
 5. The method of claim 1, wherein the first allele of the gene ofthe first livestock breed comprises a modification, relative to thesecond allele of the gene, that is chosen from the group consisting ofan insertion, a deletion, a polymorphism, and a single nucleotidepolymorphism (SNP).
 6. The method of claim 1, wherein the second alleleof the gene comprises a myostatin allele present in Belgian Blue cattle.7. The method of claim 1, wherein said introducing of the TALENcomprises introducing an mRNA encoding the TALEN or introducing a vectorcomprising a nucleic acid sequence encoding the TALEN.
 8. The method ofclaim 7, further comprising introducing into the cell or embryo a vectorcomprising a reporter gene, wherein introducing the vector comprisingthe report gene is independent of said introducing of the TALEN.
 9. Amethod of producing a live gene-edited porcine or bovine animalcomprising: (a) providing: (i) a primary somatic cell or an embryo froma porcine or bovine animal comprising in its genome a first allele of agene; (ii) a transcription activator like effector fused to anendonuclease (TALEN) comprising an ability to target, bind, and cleaveDNA in a specific site of the first allele of the gene to create adouble-stranded break in the DNA at the specific site; and (iii) ahomology directed repair (HDR) template comprising an exogenous nucleicacid sequence encoding a second allele of the gene flanked by DNAsequences that are homologous to the first allele of the gene; (b)introducing the TALEN and the HDR template into the primary somatic cellor the embryo, wherein the TALEN creates a double-stranded break in theDNA at the specific site in the first allele of the gene in the cell orembryo and the HDR template is inserted into the double-stranded breakin the DNA; thereby producing an gene-edited porcine or bovine primarysomatic cell or embryo; and (c) producing a gene-edited porcine orbovine animal by (i) introducing the gene-edited porcine or bovineprimary somatic cell into an enucleated porcine or bovine oocyte toproduce a nuclear transfer embryo and transferring the nuclear transferembryo into a surrogate female to produce a live gene-edited porcine orbovine animal; or (ii) transferring the gene-edited porcine or bovineembryo into a surrogate female to produce a live gene-edited porcine orbovine animal.
 10. The method of claim 9, wherein said introducing ofthe TALEN comprises introducing an mRNA encoding the TALEN.
 11. Themethod of claim 9, wherein the method is performed without exposing theprimary somatic cell or the embryo from the porcine or bovine animal toa reporter gene that, if incorporated into the chromosomal DNA of theprimary somatic cell or the embryo confers a trait on the primarysomatic cell or the embryo that permits isolation by survival selectioncriterion.
 12. The method of claim 9, wherein the second allele is anatural allele of a different breed, a different species, or a differentlineage than the primary somatic cell or the embryo provided in step(a)(i).
 13. The method of claim 9, wherein the live gene-edited porcineor bovine animal is heterozygous for the second allele.
 14. The methodof claim 9, wherein the live gene-edited porcine or bovine animal ishomozygous for the second allele.
 15. The method of claim 9, wherein thefirst allele is a natural allele from a first breed and the secondallele is a natural allele from a second breed.
 16. The method of claim9, wherein the primary somatic cell or the embryo is from a bovineanimal.
 17. The method of claim 15, wherein the first breed is a Wagyuor Nelore cattle and the second breed is a Belgian Blue cattle.
 18. Themethod of claim 9, wherein the primary somatic cell or the embryo isfrom a porcine animal.
 19. The method of claim 9, wherein theendonuclease is FokI or a portion thereof.
 20. The method of claim 9,wherein the second allele has, relative to the first allele, a featureselected from the group consisting of an insertion and a deletion. 21.The method of claim 9, wherein the second allele has, relative to thefirst allele, a single-nucleotide polymorphism (SNP).
 22. The method ofclaim 9, wherein said introducing of the TALEN comprises introducing aplasmid DNA that is transcribed into an mRNA encoding the TALEN.